Investigating the Mechanism of Horseradish Peroxidase As a RAFT-Initiase

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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 S M M S Mj + Mk Mj + k j Mk 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 initiation of polymerization, and bottom: RAFT equilibrium and degenerative transfer. bottom: RAFT equilibrium and degenerative transfer. The advantages of HRP-catalyzed RAFT polymerization can be optimized through a more Thethorough advantages understanding of HRP-catalyzed of its reaction RAFTprocesses. polymerization The specific mechanism can be optimized and reaction through kinetics aof more thorough understanding of its reaction processes. The speciﬁc mechanism and reaction kinetics of HRP-catalyzed polymerization have yet to be fully explored and are the focus of this study. Zhang et al. reported pseudo-ﬁrst order kinetics, which are observed due the semilogarithmic conversion of Polymers 2018, 10, 741 3 of 14 polymer being linearly related to time [41]. Previous studies have focused on the kinetics of HRP assays and have demonstrated the inhibitory effects of hydrogen peroxide on HRP activity [45,48]. Our earlier study included a brief report of how HRP activity is affected by the radical transfer components CTA and ACAC when included in the assay. However, the scope of the previous study was limited in that all HRP-catalyzed polymerizations were run under the same conditions and the effect of altering reaction conditions such as reactant concentration was not investigated. This study examines how the reaction rate of HRP-catalyzed RAFT polymerization is affected when the reaction components HRP, CTA, hydrogen peroxide, and ACAC are varied. In this work, a detailed kinetic study is performed in each component of the HRP catalyzed RAFT process. The target of this work is to correlate the observed polymerization reaction kinetics to the underlying enzymatic activity, guiding how to optimize the HRP-catalyzed RAFT polymerization.

2. Experimental

2.1. Materials All materials were purchased from commercial suppliers unless otherwise speciﬁed. All materials were used as received unless otherwise speciﬁed. 2,4-pentadione or acetylacetone (Alfa Aesar, acac, Tewksbury, MA, USA) was used as received. N,N-dimethylacrylamide (DMAm, Acros Organics, Tewksbury, MA, USA) was passed over a short column of basic alumina to remove the inhibitor. Horseradish peroxidase type I powder (HRP, 146 units/mg, Sigma, Burlington, VT, USA) was stored at 4 ◦C. (2-propionic acid)yl ethyl trithiocarbonate (PAETC) was synthesized following procedures described in the literature [49,50].

2.2. Typical HRP Catalyzed RAFT Polymerization of DMAm A clean 10 mL round bottom ﬂask was used as the reaction vessel. DMAm (72.2 mg, 729 µmol), a stock solution of PAETC in 20 mM acetate buffer at pH = 5.5 (1 mL, 1.53 mg PAETC, 7.29 µmol PAETC), and pH 5.5, 20 mM acetate buffer (3 mL) were added initially. This solution was deoxygenated by bubbling nitrogen gas through it for 10 min. A 2.7% solution of H2O2 (14 µL, 0.42 mg H2O2, 12.3 µmol H2O2) and ACAC (7 µL, 6.9 mg ACAC, 69 µmol ACAC) were added to the reaction solution. A 15 mg/mL stock solution of HRP was then prepared in pH 5.5, 20 mM acetate buffer. 200 µL of this HRP solution was added to the reaction mixture. The reaction mixture was gently stirred at 25 ◦C and samples of approximately 100 µL were taken periodically to monitor the polymerization progress. Each sample was immediately exposed to oxygen and then frozen in liquid nitrogen to terminate any polymerization progress. The samples were analyzed after thawing. NMR analysis of samples was conducted by transferring 25–40 µL of sample to approximately 0.5 mL of D2O. NMR was used to measure monomer conversion using D2O as solvent. GPC analysis of samples was conducted by transferring 25–40 µL of sample to 2 mL of DMF + 0.1% LiBr. Typical variations included changing the concentration of HRP, H2O2, PAETC, and ACAC.

2.3. Typical HRP Activity Assay The activity of HRP was determined using a method adapted from the literature [48]. A 4-aminoantipyrine solution (AAP)/phenol (PhOH) working solution was prepared with 10 mL of 0.1% AAP (1 mg/mL, in water), 20 mL 0.1% PhOH solution (1 mg/mL in water), and 70 mL 20 mM phosphate buffer, pH = 6. A 14.7 mM H2O2 stock solution was prepared. A stock solution of 20 µg/mL HRP in 20 mM phosphate buffer, pH = 6 was prepared. 900 µL of working solution of AAP and PhOH, 50 µL of 0.0147 M H2O2 solution, and 50 µL of 20 µg/mL HRP solution were added to a cuvette. The absorbance change was then measured at 500 nm over 30 s and a slope was recorded in absorbance/min. Typical variations included different loadings of HRP, PAETC, H2O2, and ACAC. Polymers 2018, 10, 741 4 of 14

2.4. NMR Polymers 2018, 10, x FOR PEER REVIEW 4 of 14 All nuclear magnetic resonance (NMR) was performed on a Bruker 500 MHz spectrometer (Billerica,2.4. NMR MA, USA). All nuclear magnetic resonance (NMR) was performed on a Bruker 500 MHz spectrometer 2.5. UV-Visible Spectroscopy (Billerica, MA, USA). HRP activity assays were measured on a Spectronic Genesys 5 spectrophotometer (Waltham, MA, USA),2.5. taking UV-Visible measurements Spectroscopy at 500 nm. HRP activity assays were measured on a Spectronic Genesys 5 spectrophotometer (Waltham, 2.6. SizeMA, ExclusionUSA), taking Chromatography measurements (SEC) at 500 nm. Size exclusion chromatography (SEC) was performed on an Agilent SEC system (Waldbronn, 2.6. Size Exclusion Chromatography (SEC) Germany) comprised of an Agilent 1260 isocratic pump, an Agilent autosampler, 1 × Agilent PolarGel-M-guardSize exclusion and chromatography 2 × Agilent PolarGel-M (SEC) was performed analytical on columns an Agilent and SEC an system Agilent (Waldbronn, 1260 refractive indexGermany) (RI) detector. comprisedN,N-dimethylformamide of an Agilent 1260 isocratic (DMF) withpump, 0.1 an wt %Agilent LiBr wasautosampler, the eluent 1 at× aAgilent flow rate of PolarGel-M-guard and 2 × Agilent PolarGel-M analytical columns and an Agilent 1260 refractive 1 mL/min, maintained at 50 ◦C. The system was calibrated with poly(methyl methacrylate) (PMMA) index (RI) detector. N,N-dimethylformamide (DMF) with 0.1 wt % LiBr was the eluent at a flow rate standardsof 1 mL/min, with molecular maintained weights at 50 ° theC. The range system of 617,500 was calibrated to 1010. Eachwith samplepoly(methyl was methacrylate) filtered through a PTFE(PMMA) 200 nm standards filter. with molecular weights the range of 617,500 to 1010. Each sample was filtered through a PTFE 200 nm filter. 3. Results 3.The Results focus of this study is to probe the underlying reaction mechanism in RAFT polymerization using HRPThe as focus a RAFT-initiase. of this study is A to combination probe the underlying of polymerization reaction mechanism kinetic analysis in RAFT as polymerization well as enzymatic assaysusing was HRP used as to a probe RAFT-initiase. the underlying A combination reaction process, of polymerization and to provide kinetic guidelines analysis foras optimizationwell as and implementationenzymatic assays was in future used to studies. probe the underlying reaction process, and to provide guidelines for optimizationSince this and process implementation is enzymatically in future studies. initiated, the impact of the horseradish peroxidase Since this process is enzymatically initiated, the impact of the horseradish peroxidase concentration was initially investigated. As expected in an enzymatically-catalyzed process, higher concentration was initially investigated. As expected in an enzymatically-catalyzed process, higher concentrationconcentration of enzymeof enzyme led led to to increased increased reactionreaction rates. rates. Figure Figure 1A1A shows shows linear linear semi-logarithmic semi-logarithmic plotsplots at each at each HRP HRP concentration concentration with with thethe inductioninduction decreasing decreasing with with the thehigher higher enzyme enzyme loadings. loadings. TheThe final final conversion conversion at at lower lower HRP HRP concentrationsconcentrations was was lower lower as as well. well. Figure Figure 1B 1indicatesB indicates good good agreementagreement between between theoretical theoretical and andexperimental experimental M Mn nvaluesvalues and and acceptable acceptable molar molar mass mass disparities, disparities, (Mw/M(Mwn/Mtypicallyn typically less less than than 1.40).1.40).

4 1.5x104 3 A [HRP] =0.36 mg/mL B [HRP] =0.36 mg/mL o o [HRP] =0.71 mg/mL [HRP] =0.71 mg/mL o o [HRP] =1.07 mg/mL [HRP] =1.07 mg/mL o 4 o [HRP] =1.42 mg/mL 1x10 [HRP] =1.42 mg/mL 2.5 3 o o Theory n

n 3

5x10 2 /M

2 M w M

-ln(1 -conversion) -ln(1 1 0 1.5

0 1 0 1020304050 0 0.2 0.4 0.6 0.8 1 conversion time (min)

Figure 1. (A) Semilogarithmic plots and (B) evolution of Mn (solid points) and Mw/Mn (open points) Figure 1. (A) Semilogarithmic plots and (B) evolution of Mn (solid points) and Mw/Mn (open points) of HRP-catalyzed polymerization. Red points (circles) show [HRP]0 = 0.36 mg/mL. Blue points of HRP-catalyzed polymerization. Red points (circles) show [HRP]0 = 0.36 mg/mL. Blue points (squares) show [HRP]0 = 0.71 mg/mL. Orange points (triangles) show [HRP]0 = 1.07 mg/mL. Green (squares) show [HRP]0 = 0.71 mg/mL. Orange points (triangles) show [HRP]0 = 1.07 mg/mL. Green points (diamonds) show [HRP]0 = 1.42 mg/mL. Assays were conducted with component ratios of points (diamonds) show [HRP]0 = 1.42 mg/mL. Assays were conducted with component ratios of [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:9.7:1.7, and concentrations of [DMAm] = 170 mM, [DMAm][HRP]0 :[PAETC]= X mg/mL0 :[ACAC]in 4.2 mL 0of:[H pH2O = 25.5,]0 =20100:1:9.7:1.7, mM acetate buffer and at concentrations 25 °C. of [DMAm] = 170 mM, [HRP] = X mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 ◦C.

Polymers 2018, 10, 741 5 of 14 Polymers 2018, 10, x FOR PEER REVIEW 5 of 14

AA key key component component of of enzyme-initiated enzyme-initiated RAFT RAFT is is the the chain chain transfer transfer agent agent (CTA), (CTA), for for which which concentrationconcentration is is an an important important parameter parameter to to be be inve investigated.stigated. Figure Figure 2A2 Ashows shows the the reaction reaction went went to fullto fullconversion conversion in inunder under ten ten minutes minutes when when [DMAM] [DMAM]0:[PAETC]0:[PAETC]0 0= =100:0.5, 100:0.5, however however when when [DMAM][DMAM]0:[PAETC]0:[PAETC]0 0= =100:2, 100:2, the the reaction reaction was was still still in inprogress progress after after 120 120 min. min. Lower Lower concentrations concentrations of CTAof CTA resulted resulted in ina less a less controlled controlled polymerization, polymerization, as asshown shown in in Figure Figure 2B.2B. Agreement between theoreticaltheoretical and and experimental experimental M Mn valuesn values was was especial especiallyly poor poor when when [DMAM] [DMAM]0:[PAETC]0:[PAETC]0 = 100:0.5.0 = 100:0.5. This effectThis effectis magnified is magniﬁed further further when whenconsidering considering the system the system with even with evenlower lowerCTA CTAloading, loading, i.e., [DMAM]i.e., [DMAM]0:[PAETC]0:[PAETC]0 = 100:0.25,0 = 100:0.25 which, whichis given is in given Figure in S1. Figure This S1. poorer This control poorer is control correlated is correlated with the increasedwith the increasedreaction rates, reaction and rates,could andbe expected could be if expected the system if thewith system low CTA with loading low CTA had loading too high had a radicaltoo high concentration a radical concentration for the amount for theof the amount CTA, ofwh theich CTA, is the which controlling is the agent controlling in RAFT agent systems. in RAFT It issystems. important It is to important note thatto in note an ideal that inRAFT an ideal polymerization, RAFT polymerization, the concentration the concentration of the chain of thetransfer chain agenttransfer should agent not should impact not the impact rate theof the rate reaction, of the reaction, since the since process the process is a dege is anerative degenerative transfer. transfer. This suggestsThis suggests that the that CTA theCTA could could be decreasing be decreasing the thepolymerization polymerization rate rate through through an an inhibitory inhibitory process process withwith the the HRP HRP enzyme. enzyme. This This will will be be further further probed probed when when evaluating evaluating enzymatic enzymatic activity. activity.

5 3x104 A [DMAM] :[PAETC] = 100:0.5 o o B [DMAM] :[PAETC] = 100:1 o o 4 [DMAM] :[PAETC] = 100:2 3 o o [DMAM] :[PAETC] = 100:0.5 4 o o [DMAM] :[PAETC] = 100:1 2x10 o o [DMAM] :[PAETC] = 100:2 3 o o

Theory n n /M w M 4

2 1x10 2 M

-ln(1 -conversion) -ln(1 1 0

0 1 0 20406080100120140 0 0.2 0.4 0.6 0.8 1 time (min) conversion

FigureFigure 2. 2. (A(A)) Semilogarithmic Semilogarithmic plots plots and and ( (BB)) evolution evolution of Mn (solid(solid points) and MMw/M/Mn n(open(open points) points) ofof HRP-catalyzed polymerization.polymerization. Red Red points points (circles) (circles) show show[DMAM] [DMAM]0:[PAETC]0:[PAETC]0 = 100:0.50 = 100:0.5.. Blue pointsBlue points(squares) (squares) show [DMAM] show0:[PAETC] [DMAM]0 = 100:1.0:[PAETC] Green0 points= 100:1. (diamonds) Green show points [DMAM] (diamonds)0:[PAETC] 0 =show 100:2. [DMAM]Assays were0:[PAETC] conducted0 = with 100:2. component Assays ratios ofwere[DMAm] conducted0:[PAETC] 0with:[ACAC] component0:[H2O2]0 = 100:ratiosX:9.7:1.7 of , [DMAm]and concentrations0:[PAETC]0:[ACAC] of [DMAm]0:[H =2O 1702]0 mM= 100:, [HRP]X:9.7:1.7, = 0.71 and mg/mL concentrationsin 4.2 mL ofof pH [DMAm] = 5.5, 20 = mM 170 acetate mM, [HRP]buffer = at 0.71 25 ◦mg/mLC. in 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 °C.

HydrogenHydrogen peroxide peroxide was was then then studied studied as asa reacti a reactionon parameter. parameter. Since Since hydrogen hydrogen peroxide peroxide is the is substratethe substrate for HRP, for HRP,a higher a higher concentration concentration of hydrog of hydrogenen peroxide peroxide should shouldresult in result a higher in a rate higher of radicalrate of generation, radical generation, which should which lead should to a higher lead reaction to a higher rate under reaction typical rate RAFT under polymerization typical RAFT conditions.polymerization However, conditions. hydrogen However, peroxide hydrogenis shown to peroxide be inhibitory is shown to HRP to beat higher inhibitory concentrations to HRP at (Figureshigher concentrations3A and 7), which (Figures results3A in and reduced 7 ), which polyme resultsrization in reducedreaction polymerizationrates. Figure 3A reactionshows linear rates. conversionFigure3A shows at lineareach conversionhydrogen atperoxide each hydrogen concentration. peroxide concentration. Conversion Conversionwas reduced was reducedwhen [PAETC]0:[H2O2] = 1:0.59. Figure 3B indicates acceptable agreement between theoretical and when [PAETC]0:[H2O2] = 1:0.59. Figure3B indicates acceptable agreement between theoretical and experimental Mn values and acceptable molar mass disparities, (Mw/Mn typically less than 1.30). The experimental Mn values and acceptable molar mass disparities, (Mw/Mn typically less than 1.30). complexThe complex behavior behavior of polymerization of polymerization rate with rate withhydrogen hydrogen peroxide peroxide concentration concentration suggests suggests a dual a role dual for this reagent, one as a substrate and the other as an inhibitor or denaturant of the enzyme. This role for this reagent, one as a substrate and the other as an inhibitor or denaturant of the enzyme. will be investigated in greater detail when probing the underlying enzymatic activity. This will be investigated in greater detail when probing the underlying enzymatic activity.

Polymers 2018, 10, 741 6 of 14 Polymers 2018, 10, x FOR PEER REVIEW 6 of 14

Polymers 2018, 10, x FOR PEER REVIEW 6 of 14 4 1.5x104 3 A [PAETC] :[H O ]= 1:0.59 o 2 2 B [PAETC] :[H O ]= 1:0.59 [PAETC] :[H O ]= 1:0.85 o 2 2 o 2 2 [PAETC] :[H O ]= 1:0.85 4 4 o 2 2 [PAETC] :[H O ]= 1:1.69 1.5x10 3 o 2 2 [PAETC] :[H O ]= 1:1.69 A [PAETC] :[H O ]= 1:0.59 o 2 2 3 o 2 2 B [PAETC] :[H O ]= 1:0.59 [PAETC] :[H O ]= 1:2.53 4 [PAETC]o:[H2O2]= 1:2.53 2.5 [PAETC]o:[H2O2]= 1:0.85 1x10 o 2 2 o 2 2 [PAETC]Theory :[H O ]= 1:0.85 [PAETC] :[H O ]= 1:1.69 o 2 2 o 2 2 [PAETC] :[H O ]= 1:1.69 3 o 2 2 [PAETC] :[H O ]= 1:2.53 4 [PAETC] :[H O ]= 1:2.53 2.5 n 2 o 2 2 1x10 o 2 2 /M n Theory 2 3 w M 5x10 M n 2 /M n 2 3 w M -ln(1 -conversion) 1 5x10 1.5 M 0

-ln(1 -conversion) 1 1.5 0 0 1 0 1020304050 0 0.2 0.4 0.6 0.8 1 time (min) conversion 0 1 0 1020304050 0 0.2 0.4 0.6 0.8 1 Figure 3. (A) Semilogarithmic plots and (B) evolution of Mn (solid point) and Mw/Mn (open points) of Figure 3. (A) Semilogarithmictime (min) plots and (B) evolution of Mn (solid point)conversion and Mw/Mn (open points) of HRP-catalyzed polymerization. Red points (circles) show [PAETC]0:[H2O2] = 1:0.59. Purple points HRP-catalyzed polymerization. Red points (circles) show [PAETC]0:[H2O2] = 1:0.59. Purple points Figure(triangles) 3. (A show) Semilogarithmic [PAETC]0:[H plots2O2] =and 1:0.85. (B) evolution Blue points of M (squares)n (solid point) show and [PAETC] Mw/Mn0 :[H(open2O2 ]points) = 1:1.69. of (triangles) show [PAETC]0:[H2O2] = 1:0.85. Blue points (squares) show [PAETC]0:[H2O2] = 1:1.69. HRP-catalyzedGreen points (diamonds)polymerization. show Red [PAETC] points (circles)0:[H2O2] show= 1:2.53. [PAETC] Component0:[H2O2] = ratios1:0.59. utilizedPurple pointswere Green points (diamonds) show [PAETC]0:[H2O2] = 1:2.53. Component ratios utilized were (triangles)[DMAm]0:[PAETC] show [PAETC]0:[ACAC]0:[H0:[H2O22O] =2] 0 1:0.85.= 100:1:9.7: Blue Xpoints, and concentrations(squares) show were [PAETC] [DMAm]0:[H2O =2 ] 170= 1:1.69. mM, [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:9.7:X, and concentrations were [DMAm] = 170 mM, Green[HRP] =points 0.71 mg/mL (diamonds) in 4.2 mL show of pH [PAETC] = 5.5, 200 mM:[H2O acetate2] = 1:2.53. buffer atComponent 25 °C. ratios utilized were [HRP] = 0.71 mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 ◦C. [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:9.7:X, and concentrations were [DMAm] = 170 mM, [HRP]ACAC = was0.71 mg/mLthe final in reaction 4.2 mL of parameter pH = 5.5, 20 investigated mM acetate buffer as related at 25 °C.to kinetics. ACAC acts primarily asACAC a mediator, was thetransferring ﬁnal reaction the OH parameter and heme investigated bound radicals as related generated to kinetics. by HRP ACACto simple acts carbon primarily as acentered mediator,ACAC radicals was transferring the that final are reaction capable the OH parameter of and initiating heme investigated boundpolymerization. radicals as related Figure generated to kinetics. 4A indicates by ACAC HRP linear acts to simple primarily reaction carbon centeredasprogress, a mediator, radicals with transferringsome that aredeviation capable the OHwhen of and initiating [PAETC] heme bound0 polymerization.:[ACAC] radicals = 1:7.10. generated No Figure reaction by4A HRP indicates was to observed simple linear carbon when reaction centered[ACAC] =radicals 0 mM, thatwhich are shows capable that of it initiating is an essential polymerization. component Figure to HRP-catalyzed 4A indicates polymerization. linear reaction progress, with some deviation when [PAETC]0:[ACAC] = 1:7.10. No reaction was observed when progress,Further, the with reaction some ratedeviation increased, when albeit [PAETC] in a diminishing0:[ACAC] = fashion,1:7.10. No at hireactiongher ACAC was observedconcentrations. when [ACAC] = 0 mM, which shows that it is an essential component to HRP-catalyzed polymerization. [ACAC]The kinetic = 0 datamM, suggestwhich shows that there that itis is a ancritical essential concentration component of to the HRP-catalyzed mediator ACAC polymerization. needed for Further, the reaction rate increased, albeit in a diminishing fashion, at higher ACAC concentrations. Further,efficient thepolymerization. reaction rate increased,In addition, albeit control in a diminishingover the polymerization fashion, at hi gherwas ACACreduced concentrations. when ACAC TheTheconcentrations kinetic kinetic data data suggest are suggest lowered, that that there as thereshown is a is critical in a Figurecritical concentration 4B. concentration There is ofreducedthe of mediatorthe agreement mediator ACAC betweenACAC needed needed theoretical for efﬁcientfor polymerization. In addition, control over the polymerization was reduced when ACAC concentrations efficientand experimental polymerization. Mn values In whenaddition, [PAETC] control0:[ACAC] over the = 1:7.10. polymerization was reduced when ACAC areconcentrations lowered, as shown are lowered, in Figure as 4shownB. There in Figure is reduced 4B. There agreement is reduced between agreement theoretical between and theoretical experimental and experimental Mn values when [PAETC]0:[ACAC] = 1:7.10. Mn values3.5 when [PAETC]0:[ACAC] = 1:7.10. 3 [PAETC] :[ACAC]= 1:7.10 B [PAETC] :[ACAC]= 1:7.10 o A o 4 [PAETC] :[ACAC]= 1:9.46 [PAETC] :[ACAC]= 1:9.46 o 1.5x10 o 3 [PAETC] :[ACAC]= 1:18.93 3 3.5 [PAETC] :[ACAC]= 1:18.93 o [PAETC]o:[ACAC]= 1:7.10 B [PAETC] :[ACAC]= 1:7.10 A o Theory o 2.5 4 [PAETC] :[ACAC]= 1:9.46 2.5 [PAETC] :[ACAC]= 1:9.46 1.5x10 o 3 o [PAETC] :[ACAC]= 1:18.93 4 [PAETC] :[ACAC]= 1:18.93 o o 1x10 Theory 2 2.5 n

2.5 n 2 /M w M 1x104

1.5 M 2 3 n

n 5x10 2 /M w M

-ln(1 -conversion) -ln(1 1

1.5 1.5 M 5x103 0.5

-ln(1 -conversion) -ln(1 1 0 1.5 0 0.5 010203040 1 0 0 0.2 0.4 0.6 0.8 1 time (min) conversion 0 1 010203040 0 0.2 0.4 0.6 0.8 1 Figure 4. (A) Semilogarithmic plots and (B) evolution of Mn (solid point) and Mw/Mn (open points) of time (min) conversion HRP-catalyzed polymerization. Red points (circles) show [PAETC]0:[ACAC] = 1:7.10. Blue points

Figure(squares) 4. (Ashow) Semilogarithmic [PAETC]0:[ACAC] plots and= 1:9.46. (B) evolution Green pointsof Mn (solid(diamonds) point) andshow M w[PAETC]/Mn (open0:[ACAC] points) of = Figure 4. (A) Semilogarithmic plots and (B) evolution of Mn (solid point) and Mw/Mn (open points) HRP-catalyzed1:18.93. Component polymerization. ratios utilized Red werepoints [DMAm] (circles)0 :[PAETC]show [PAETC]0:[ACAC]0:[ACAC]0:[H2O 2=]0 1:7.10.= 100:1: BlueX:1.7, points and of HRP-catalyzed polymerization. Red points (circles) show [PAETC]0:[ACAC] = 1:7.10. Blue points (squares)concentrations show were [PAETC] [DMAm]0:[ACAC] = 170 =mM, 1:9.46. [HRP] Green = 0.71 points mg/mL (diamonds) in 4.2 mL showof pH [PAETC]= 5.5, 20 mM0:[ACAC] acetate = (squares) show [PAETC]0:[ACAC] = 1:9.46. Green points (diamonds) show [PAETC]0:[ACAC] = 1:18.93. 1:18.93.buffer at Component 25 °C. ratios utilized were [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:X:1.7, and Component ratios utilized were [DMAm] :[PAETC] :[ACAC] :[H O ] = 100:1:X:1.7, and concentrations concentrations were [DMAm] = 170 mM,0 [HRP] = 0.710 mg/mL0 in 24.22 mL0 of pH = 5.5, 20 mM acetate were [DMAm] = 170 mM, [HRP] = 0.71 mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 ◦C. buffer at 25 °C.

Polymers 2018, 10, 741 7 of 14 Polymers 2018, 10, x FOR PEER REVIEW 7 of 14

InIn order order to to investigate investigate whether whether the the observed observed kine kinetictic trends trends are are due due to to impacts impacts of of the the reagent reagent on on thethe HRPHRP enzymatic enzymatic turn turn over, over, or due or to due down-stream to down effects-stream on effects the RAFT on process, the RAFT the polymerization process, the polymerizationkinetics were correlated kinetics were with correlated the intrinsic with enzymatic the intrinsic assay enzymatic kinetics assay as a function kinetics ofas eacha function reaction of eachcomponent. reaction Initially, component. the impact Initially, of enzymethe impact loading of enzyme was investigated. loading was Figure investigated. S2 indicates Figure that theS2 indicatesaddition ofthat DMAm the addition monomer of at DMAm the concentrations monomer at used thein concentrations these reactions used hasnegligible in these reactions impact on has the negligibleenzymatic impact activity. on As the shown enzymatic in Figure activity.5, increased As shown enzyme in Figure loading 5, ledincreased to a higher enzyme apparent loading enzymatic led to a app higheractivity, apparent and this enzymatic correlated activity, well to and the this apparent correla rateted well of polymerization to the apparent (k ratep of). polymerization As anticipated, (kthispapp shows). As anticipated, that, as anticipated, this shows higher that, enzymeas anticipa concentrationsted, higher enzyme lead to concentrations an increase in lead the rateto an of increaseradical production, in the rate leadingof radical to bothproduction, a faster leading rate of polymerization, to both a faster and rate a of higher polymerization, rate of turnover and ofa higherphenol/aminoantipyrine rate of turnover of tophenol/aminoantipyrine the products. to the products.

A 1.2 B 0.35 1 0.3 0.25

0.8 ) -1 0.2

0.6 (min 0.15 app p

0.4 k 0.1

Normalized Activity Normalized 0.2 0.05

0 0 0 25 50 75 100 00.511.5 [HRP] (mg/mL) [HRP] (mg/L) FigureFigure 5. 5. (A(A) )Relative Relative rate rate of of enzymatic enzymatic activity, activity, as as a a function function of of HRP HRP loading, loading, measured measured by by the the reactionreaction ofof aminoantipyrineaminoantipyrine and and phenol, phenol, normalized normalized to activity to activity at 100 mg/Lat 100 HRP mg/L loading, HRP (loading,B) Apparent (B) app app Apparentpolymerization polymerization rate (kp )rate under (kp the) conditions:under the [DMAm]conditions:0:[PAETC] [DMAm]0:[ACAC]0:[PAETC]0:[H02:[ACAC]O2]0 = 100:1:9.7:1.70:[H2O2]0 = , 100:1:9.7:1.7,[DMAm] = 170 [DMAm] mM, [HRP] = 170 = mM,X mg/mL [HRP] in = 4.2X mg/mL mL of pHin 4.2 = 5.5, mL 20 of mM pH acetate= 5.5, 20 buffer mM acetate at 25 ◦C. buffer at 25 °C. Interestingly, when considering the chain transfer agent, PAETC, the polymerization kinetics in Interestingly, when considering the chain transfer agent, PAETC, the polymerization kinetics in Figure2 indicated a decrease in rate. To determine the origin of this reduction in polymerization rate, Figure 2 indicated a decrease in rate. To determine the origin of this reduction in polymerization HRP enzymatic assays were performed in conjunction with the polymerization results. As indicated in rate, HRP enzymatic assays were performed in conjunction with the polymerization results. As Figure6, low concentrations of PAETC had minimal impact on the enzymatic activity or polymerization indicated in Figure 6, low concentrations of PAETC had minimal impact on the enzymatic activity or rate, however, higher concentrations of the PAETC RAFT agent led to substantial decreases in the polymerization rate, however, higher concentrations of the PAETC RAFT agent led to substantial enzymatic activity, both as the ability to react phenol with aminoantipyrine as well as the enzyme’s decreases in the enzymatic activity, both as the ability to react phenol with aminoantipyrine as well ability to act as a RAFT initiase. These data suggest that the PAETC is perturbing the enzyme, possibly as the enzyme’s ability to act as a RAFT initiase. These data suggest that the PAETC is perturbing the at the active site, decreasing its ability to act as an initiator for RAFT polymerization. Nevertheless, enzyme, possibly at the active site, decreasing its ability to act as an initiator for RAFT non-zero polymerization rate and enzymatic activity is observed in all cases studied. This indicates polymerization. Nevertheless, non-zero polymerization rate and enzymatic activity is observed in all that molecular weight can be effectively controlled by the enzymatic RAFT process, although lower cases studied. This indicates that molecular weight can be effectively controlled by the enzymatic targeted molecular weights will typically lead to slower polymerizations. RAFT process, although lower targeted molecular weights will typically lead to slower The substrate of HRP is H2O2, which would intuitively suggest that higher loadings of H2O2 polymerizations. should lead to increased enzymatic activity and polymerization. However, as displayed in Figure7, there is an optimal loading of H2O2 near 0.2–0.4 mM in the enzymatic assays and 1 mM in the polymerization experiments, that gives highest performance of the enzyme. At low H2O2 loading the turnover rate is low, presumably due to the low substrate concentration, while at high H2O2 loading the enzyme could be deactivated by the highly reactive H2O2 substrate [48]. This indicates that there is an optimal concentration of H2O2 that gives a balance between enzymatic stability and performance with loading of the peroxide substrate.

Polymers 2018, 10, x FOR PEER REVIEW 8 of 14

A 1.2 B 0.6 1 0.5

0.8 ) 0.4 -1

0.6 0.3 Polymers 2018, 10, 741 (min 8 of 14 app

Polymers 2018, 10, x FOR PEER REVIEW p 8 of 14

0.4 k 0.2

Normalized Activity Normalized 1.2 A 0.2 B 0.60.1

10 0.50 0 0.7704 1.557 3.114 01234

0.8 [PAETC] (mM) ) 0.4 [PAETC] (mM)

-1 Figure0.6 6. (A) Relative rate of enzymatic activity, as a function0.3 of PAETC loading, measured by the (min reaction of aminoantipyrine and phenol, normalized to activity at no PAETC loading; (B) Apparent app p papp 0 0 0 2 2 0 polymerization0.4 rate (k ) under the conditions:k 0.2[DMAm] :[PAETC] :[ACAC] :[H O ] = 100:X:9.7:1.7, [DMAm] = 170 mM, [HRP] = 0.71 mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 Activity Normalized 0.2°C. 0.1

The 0substrate of HRP is H2O2, which would intuitively0 suggest that higher loadings of H2O2 should lead to increased0 0.7704 enzymatic 1.557 activity 3.114 and polymerization.01234 However, as displayed in Figure 7,

[PAETC] (mM)2 2 there is an optimal loading of H O near 0.2–0.4 mM in the enzymatic[PAETC] assays (mM) and 1 mM in the polymerization experiments, that gives highest performance of the enzyme. At low H2O2 loading the Figure 6. (A) Relative rate of enzymatic activity, as a function of PAETC loading, measured by the turnoverFigure 6. rate(A )is Relative low, presumably rate of enzymatic due to the activity, low substrate as a function concentration, of PAETC while loading, at high measured H2O2 loading by the reaction of aminoantipyrine and phenol, normalized to activity at no PAETC loading; (B) Apparent thereaction enzyme of aminoantipyrinecould be deactivated and phenol,by the highly normalized reactive to H activity2O2 substrate at no PAETC [48]. This loading; indicates (B) Apparentthat there polymerization rateapp (kpapp) under the conditions: [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = ispolymerization an optimal rateconcentration (kp ) under of theH2 conditions:O2 that gives[DMAm] a balance0:[PAETC] between0:[ACAC] enzymatic0:[H2O2]0 =stability 100:X:9.7:1.7 and, 100:X:9.7:1.7, [DMAm] = 170 mM, [HRP] = 0.71 mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer at performance[DMAm] = 170 with mM, loading [HRP] of = 0.71the peroxide mg/mL in substrate. 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 ◦C. 25 °C.

1.5 AThe substrate of HRP is H2O2, which would intuitivelyB 0.25 suggest that higher loadings of H2O2 should lead to increased enzymatic activity and polymerization. However, as displayed in Figure 7, there is an optimal loading of H2O2 near 0.2–0.4 mM 0.2in the enzymatic assays and 1 mM in the polymerization experiments, that gives highest performance of the enzyme. At low H2O2 loading the 1 turnover rate is low, presumably due to the low substrate) concentration, while at high H2O2 loading -1 0.15 the enzyme could be deactivated by the highly reactive H2O2 substrate [48]. This indicates that there

is an optimal concentration of H2O2 that gives (min a balance between enzymatic stability and

performance with loading of the peroxide substrate. app 0.1 p

0.5 k

A Normalized Activity 1.5 B 0.250.05

0 0.20 0 0.07 0.18 0.37 0.74 1.47 3.68 7.35 0123456 1 [H O ] (mM) ) [H O ] (mM) 2 2 2 2 -1 0.15

Figure 7. (A) Relative rate of enzymatic activity, as a function of H2O2 loading, measured by the

Figure 7. (A) Relative rate of enzymatic activity, as a function(min of H2O2 loading, measured by the reaction reaction of aminoantipyrine and phenol, normalized to activity at [H2O2] = 0.74 mM loading; (B) of aminoantipyrine and phenol, normalized to activityapp at0.1 [H2O2] = 0.74 mM loading; (B) Apparent papp p 0 0 0 2 2 0 Apparent0.5 polymerizationapp rate (k ) under the conditions:k [DMAm] :[PAETC] :[ACAC] :[H O ] = polymerization rate (kp ) under the conditions: [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:9.7:X, 100:1:9.7:X, [DMAm] = 170 mM, [HRP] = 0.71 mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer◦ at 25

[DMAm]Normalized Activity = 170 mM, [HRP] = 0.71 mg/mL in 4.2 mL of pH = 5.5, 20 mM acetate buffer at 25 C. °C. 0.05

The ﬁnal0 parameter to be investigated is the loading0 of the ACAC mediator. Kinetic analysis in 0 0.07 0.18 0.37app 0.74 1.47 3.68 7.35 Figure8 indicates that the k p value increases and eventually0123456 plateaus when loadings of ACAC [H O ] (mM) [H O ] (mM) are increased. In contrast2 the2 apparent enzymatic activity, as measured2 by2 the rate of reaction of phenol with aminoantipyrine, decreases with increasing ACAC loading. The results in Figure8 are Figure 7. (A) Relative rate of enzymatic activity, as a function of H2O2 loading, measured by the unlike thereaction other of assays, aminoantipyrine where apparent and phenol, enzymatic normalized activity to activity correlated at [H2O2 well] = 0.74 with mM polymerization loading; (B) rate.

However,Apparent it is important polymerization to note rate that (kp ACACapp) under is athe mediator conditions: of the[DMAm] polymerization0:[PAETC]0:[ACAC] and serves0:[H2O2] to0 = generate carbon centered100:1:9.7:X radicals, [DMAm] from= 170 mM, the [HRP] highly = 0.71 reactive mg/mL hydroxyl in 4.2 mL of and pH = heme 5.5, 20 centeredmM acetate radicals buffer at 25 generated through°C. the HRP catalytic cycle. Therefore, at a sufﬁciently high ACAC loading, essentially all radicals generated by HRP will react with ACAC to create carbon centered radicals capable of initiating polymerization, leading to a plateau in rate with ACAC loading. However, by the same mechanism, at higher ACAC loadings, ACAC derived radicals will be generated, rather than the product of phenol with aminoantipyrine, leading to a decrease in apparent enzymatic activity. Therefore, the reduction in Polymers 2018, 10, x FOR PEER REVIEW 9 of 14

The final parameter to be investigated is the loading of the ACAC mediator. Kinetic analysis in Figure 8 indicates that the kpapp value increases and eventually plateaus when loadings of ACAC are increased. In contrast the apparent enzymatic activity, as measured by the rate of reaction of phenol with aminoantipyrine, decreases with increasing ACAC loading. The results in Figure 8 are unlike the other assays, where apparent enzymatic activity correlated well with polymerization rate. However, it is important to note that ACAC is a mediator of the polymerization and serves to generate carbon centered radicals from the highly reactive hydroxyl and heme centered radicals generated through the HRP catalytic cycle. Therefore, at a sufficiently high ACAC loading, essentially all radicals generated by HRP will react with ACAC to create carbon centered radicals capable of initiating polymerization, leading to a plateau in rate with ACAC loading. However, by Polymers 2018 10 the same mechanism,, , 741 at higher ACAC loadings, ACAC derived radicals will be generated, rather9 of 14 than the product of phenol with aminoantipyrine, leading to a decrease in apparent enzymatic activity.apparent Therefore, HRP activity the inreduction Figure8A in provides apparent evidence HRP activity for ACAC in Figure acting as8A a mediatorprovides inevidence enzymatic for ACACRAFT polymerization.acting as a mediator in enzymatic RAFT polymerization.

A 1.2 B 0.2 1 0.15

0.8 ) -1

0.1 0.6 (min app p

0.4 k 0.05 Normalized Activity 0.2

0 0 0 24.3 48.7 97.3 195 389 973 0 5 10 15 20 25 30 35 [ACAC] (mM) [ACAC](mM)

FigureFigure 8. 8. ((AA)) Relative Relative rate rate of of enzymatic enzymatic activity, activity, as as a a function function of of ACAC ACAC loading, loading, measured measured by by the the reactionreaction of of aminoantipyrine aminoantipyrine and and phenol, phenol, norma normalizedlized to to activity activity at at no no ACAC ACAC loading; loading; ( (BB)) Apparent Apparent polymerization rate (kpapp) under the conditions: [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:X:1.7, polymerization rate (kp ) under the conditions: [DMAm]0:[PAETC]0:[ACAC]0:[H2O2]0 = 100:1:X:1.7, [DMAm][DMAm] = = 170 170 mM, mM, [HRP] [HRP] = = 0.71 0.71 mg/mL mg/mL in in 4.2 4.2 mL mL of of pH pH = =5.5, 5.5, 20 20 mM mM acetate acetate buffer buffer at at 25 25 °C.◦C.

4.4. Discussion Discussion TheThe key key results results of of the the earlier earlier analysis analysis are are th thatat enzymatic enzymatic activity activity correlates correlates well well with with polymerizationpolymerization efficiency efﬁciency and and rate. rate. The The majority majority of of systems systems displayed displayed a a short short induction induction period, period, which could be due to residual oxygen or the catalase-like activity of HRP in the presence of the which could be due to residual oxygen or the catalase-like activity of HRP in the presence of the H2O2 Hsubstrate2O2 substrate [51]. [51]. Note Note that tothat minimize to minimize freezing freezin inducedg induced denaturation denaturation of the of enzyme the enzyme [52,53 ],[52,53], freeze freezepump pump thaw cycles thaw arecycles not are performed not performed on the enzymeon the enzyme containing containing solutions, solutions, which could which lead could to traces lead to of tracesresidual of oxygen.residual Nevertheless, oxygen. Nevertheless, rapid polymerization rapid polymerization typically occurs typically after this occurs short inductionafter this period.short inductionThis indicates period. that This polymerization indicates that kinetics polymerization are primarily kinetics dictated are by enzymaticprimarily dictated performance. by enzymatic However, performance.it is important However, to discuss eachit is resultimportant and explainto discu anyss counterintuitiveeach result and observations. explain any Ifcounterintuitive operating under observations.simple Michaelis-Menten If operating kinetics, under itsimple would Michaeli be expecteds-Menten that increasing kinetics, theit would concentration be expected of HRP that for increasingthe HRP-catalyzed the concentration polymerization of HRP should for the lead HRP-catalyzed to a square root polymerization scaling in apparent should propagation lead to a square rates rootwith scaling enzyme in loading.apparent This propagation is because rates the with linear enzyme increase loading. in radical This generation, is because wouldthe linear also increase lead to inan radical increase generation, in radical would termination, also lead leading to an to increa an overallse in radical square roottermination, scaling [leading46,54]. However,to an overall this squaretype of root trend scaling is not [46,54]. observed However, in Figure this5B, type which of trend suggests is not that observed the polymerization in Figure 5B, systemwhich suggests is more thatcomplicated the polymerization than a simple system Michaelis-Menten is more complicated kinetics than system a simple coupled Michaelis-Menten with free radical kinetics polymerization system coupledkinetics. with This free can beradical attributed polymerization to the inhibitory kinetics. effect This of can the be CTA attributed [55], since to higherthe inhibitory loadings effect of HRP of themay CTA lead [55], to a since much higher larger fractionloadings of of enzymatically HRP may lead active to a protein,much larger at the fraction same CTA of enzymatically concentration. Indeed, Figure6A show that there is a threshold ratio of CTA:HRP before any inhibition of HRP by the CTA is observed. Sulfur containing molecules are well documented inhibitors of HRP [55]. It is important to note that in all systems, the enzyme concentration is lower than the CTA concentration showing that the CTA does not quantitatively inhibit the HRP enzyme, suggesting a relatively weak or reversible mode of inhibition. When the concentrations of CTA:HRP are below that threshold ratio, no inhibition is observed. This would suggest that the trend in reaction rate vs. HRP concentration would have two distinct forms. A linear like trend similar to what is seen in Figure5B would be expected on lower HRP concentration, where the CTA:HRP ratio is higher. Concentrations of HRP greater than this would be when the CTA:HRP ratio is greater than the threshold ratio, which represents fewer HRP being inhibited by the CTA. Polymers 2018, 10, 741 10 of 14

The decreasing trend of apparent HRP activity when concentrations of ACAC are increased (as shown in Figure8A) can be explained by ACAC competing with 4-aminoantipyrine for HRP-produced radicals, which lowers the amount of oxidized 4-aminoantipyrine produced. The decrease in normalized activity corresponds to the amount of radicals reacting with ACAC [44]. These results show that increased concentrations of ACAC will produce more radicals from hydrogen peroxide, however the effect is lowered at higher concentrations. This can be explained by ACAC being in excess at these higher concentrations and is reacting with the hydrogen peroxide near its maximum rate. Figure8B shows a complementary result that, as the concentration of ACAC approaches 20 mM, there is a sufficient amount of ACAC to match the rate of HRP-catalyzed hydrogen peroxide radicals, so the overall reaction rate increases marginally despite the much higher increase in ACAC concentration. Hydrogen peroxide is known to reversibly inhibit HRP at high concentrations [48]. However, it also acts as the substrate and follows basic enzyme kinetics, to a certain extent, where increasing the substrate will lead to an increase in catalysis rate. HRP also has catalase [51], or oxygen evolving, activity against H2O2, which could decrease the rate of the reaction. Therefore, H2O2 has a competitive roles in the polymerization acting as both an inhibitor at high concentrations as well as a necessary substrate in order to generate the radicals [56]. Figure7A,B show hydrogen peroxide having both a positive and negative effect on HRP catalysis. Both the activity assay system and the HRP-catalyzed polymerization system had maximum reaction rates when hydrogen peroxide concentrations were around 0.2–1 mM, with differences likely due to the enzyme and the different ratios of H2O2 to HRP ([HRP] = 0.020 mg/mL for the activity assays and [HRP] = 0.71 mg/mL for the polymerization reaction). When considering the ratio of HRP to H2O2 where inhibition occurs, these data suggest that hydrogen peroxide has a greater inhibitory effect when HRP is acting in the polymerization system. This is most likely due to the polymerization reaction requiring a greater amount of radicals for the reaction to proceed, so increased hydrogen peroxide inhibition will decrease the reaction rate more drastically. In addition, it appears that the induction period increases with higher H2O2 concentration, possibly due to the background catalase activity of HRP [51]. Lowered concentrations of ACAC and CTA result in reduced polymerization control. This can be attributed to the role of these components in the initiation of the polymerization reaction. For example, when CTA concentration is lowered, HRP is less inhibited which results in a greater amount of radical initiator being produced. Increased initiator concentration is shown to increase the amount of terminated polymeric material, which broads the molecular weight distribution [57]. This explains the poor Mn control when [DMAM]0:[PAETC]0 = 100:0.5 in Figure2B and Figure S1 . Lowered concentrations of ACAC result in less radicals being introduced as a mediator to the polymerization reaction. This leads to insufficient initiation, which is known to result in poor polymerization control [58]. This effect is seen in Figure4B which shows poor agreement between theoretical and experimental Mn values when [PAETC]0:[ACAC] = 1:7.10. The effects of insufficient initiation are seen in Figures4A and8B as seen by the lower induction time and slightly lower apparent propagation rate. Additionally, at lower PAETC concentrations for the same H2O2 concentration there could be a non-trivial extent of RAFT agent degradation from the background reactions involving H2O2 [59]. However, it is important to note that typically elevated temperatures of 70 ◦C are typically used to remove RAFT end groups using H2O2 [59], suggesting that at the polymerization conditions minimal loss of RAFT agent should occur [36]. Considering the known catalytic cycle of HRP in the presence of H2O2 [45], a proposed cycle for initiation is developed. This is highlighted in Scheme2. The key features of this process are that initially, radicals are generated from HRP using H2O2 as a substrate. However, in the presence of ACAC, a hydrogen atom can be transferred to generate a carbon centered radical that is capable of initiating polymerization in the presence of monomer. In all cases, the role of the RAFT CTA in this mechanism is to facilitate molecular weight control through degenerative transfer, and indeed, higher CTA loadings inhibit the polymerization rate. The kinetic analysis and enzymatic assays in this work Polymers 2018, 10, x FOR PEER REVIEW 11 of 14 of 70 °C are typically used to remove RAFT end groups using H2O2 [59], suggesting that at the polymerization conditions minimal loss of RAFT agent should occur [36]. Considering the known catalytic cycle of HRP in the presence of H2O2 [45], a proposed cycle for initiation is developed. This is highlighted in Scheme 2. The key features of this process are that initially, radicals are generated from HRP using H2O2 as a substrate. However, in the presence of ACAC, a hydrogen atom can be transferred to generate a carbon centered radical that is capable of

Polymersinitiating2018 polymerization, 10, 741 in the presence of monomer. In all cases, the role of the RAFT CTA in11 ofthis 14 mechanism is to facilitate molecular weight control through degenerative transfer, and indeed, higher CTA loadings inhibit the polymerization rate. The kinetic analysis and enzymatic assays in arethis consistentwork are withconsistent the role with of HRP the role as a RAFT-initiase,of HRP as a RAFT-initiase, with molecular with weight molecular control weight enabled control by the RAFTenabled equilibrium. by the RAFT equilibrium.

Scheme 2. Proposed mechanism for enzymatic cycle in HRP-initiated RAFT polymerization.

5. Conclusions 5. Conclusions In summary, a detailed investigation into the kinetics of HRP initiated RAFT polymerization In summary, a detailed investigation into the kinetics of HRP initiated RAFT polymerization was was performed. In general, rapid and well-controlled RAFT polymerization could be performed performed. In general, rapid and well-controlled RAFT polymerization could be performed under under mild conditions near room temperature. Polymerization rate was greatly enhanced with mild conditions near room temperature. Polymerization rate was greatly enhanced with enzyme enzyme loading. The enzyme substrate H2O2 has a complex impact on polymerization rate, with an loading. The enzyme substrate H2O2 has a complex impact on polymerization rate, with an optimal optimal concentration near 1 mM under the studied conditions. A critical concentration of the concentration near 1 mM under the studied conditions. A critical concentration of the mediator, mediator, ACAC, is needed to facilitate initiation of the polymerization, although higher ACAC, is needed to facilitate initiation of the polymerization, although higher concentrations lead concentrations lead to negligible improvements in the rate of reaction. In order to have controlled to negligible improvements in the rate of reaction. In order to have controlled polymerization a polymerization a RAFT CTA is needed, although the RAFT agent used has an inhibitory effect on RAFT CTA is needed, although the RAFT agent used has an inhibitory effect on polymerization rate, polymerization rate, however, control over molecular weight is improved at higher CTA loadings. however, control over molecular weight is improved at higher CTA loadings. The observed trends in The observed trends in polymerization kinetics are rationalized through careful analysis and polymerization kinetics are rationalized through careful analysis and comparison to enzymatic activity comparison to enzymatic activity assays for HRP. assays for HRP.

Supplementary Materials:Materials:The The following following are availableare available online online at http://www.mdpi.com/2073-4360/10/7/741/s1 at www.mdpi.com/xxx/s1, Polymerization, Polymerizationkinetics with low kinetics chain transfer with low agen chaint concentrations. transfer agent concentrations.Enzymatic assay Enzymatic of HRP in assay the presence of HRP inand the absence presence of andDMAm absence monomer of DMAm. monomer.

Author Contributions: All authors contributed to the work work presented. presented. A.P.D., A.P.D., D.B.V. D.B.V.-K.,-K., J.P.B., C.T.K. designed and performed the experiments, analyzed the data. J.A.B., R.C.P. and D.K. interpreted the results and assisted and performed the experiments, analyzed the data. J.A.B., R.C.P. and D.K. interpreted the results and assisted with designing of the experiments. A.P.D. and J.A.B. assisted with the correlation of enzymatic assays and polymerizationwith designing kinetics.of the experiments. A.P.D., D.K. A.P.D. and R.C.P. and wrote J.A.B. the assisted manuscript. with the correlation of enzymatic assays and polymerization kinetics. A.P.D., D.K. and R.C.P. wrote the manuscript. Acknowledgments: This work was partially supported by the Miami University Hughes fellowships, and Start UpAcknowledgments: funding from Miami This University. work was partially supported by the Miami University Hughes fellowships, and Start ConﬂictsUp funding of from Interest: MiamiThe University. authors declare no conﬂicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. References References 1. Schmid, A.; Dordick, J.S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Industrial biocatalysis today and 1. Schmid,tomorrow. A.;Nature Dordick,2001 J.S.;, 409 Hauer,, 258–268. B.; Kiener, [CrossRef A.; ][Wubbolts,PubMed ]M.; Witholt, B. Industrial biocatalysis today and 2. tomorrow.Sheldon, R.A. Nature E factors, 2001, 409 green, 258–268. chemistry and catalysis: An odyssey. Chem. Commun. 2008, 3352–3365. [CrossRef][PubMed]

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