Article pubs.acs.org/Macromolecules
Enzyme-Initiated Reversible Addition−Fragmentation Chain Transfer Polymerization Baohua Zhang,‡ Xinjun Wang,‡ Anqi Zhu,†,‡ Kai Ma,§ Yue Lv,†,‡ Xiao Wang,†,‡ and Zesheng An*,†,‡ †
Institute of Nanochemistry and Nanobiology, ‡College of Environmental Science and Chemical Engineering, and §Department of Chemistry, Shanghai University, Shanghai 200444, China S Supporting Information *
ABSTRACT: Biocatalysis is promising for sustainable production of polymers. Enzyme-initiated reversible addition− fragmentation chain transfer (RAFT) polymerization is reported. Horseradish peroxidase (HRP) catalyzes oxidation of acetylacetone (ACAC) by hydrogen peroxide to generate ACAC radicals, which in the presence of a suitable chain transfer agent initiate efficient and well-controlled RAFT polymerization in aqueous buffer solution at room temperature. The versatility of HRP-initiated RAFT polymerization was demonstrated by controlled polymerization of a wide range of monomers, including both more and less activated monomers, under a variety of conditions, including both homogeneous solution polymerization and heterogeneous dispersion polymerization conditions. In all cases, the polymerization afforded excellent pseudo-first-order kinetics, predictable molecular weights, and narrow molecular weight distributions. Operation via RAFT mechanism of this HRP-initiated polymerization was confirmed by a combination of MALDI-ToF, NMR, and UV−vis as well as by chain extension to make well-defined block copolymers. The mildness, specificity, and biocompatibility of HRP-initiated RAFT polymerization were illustrated by controlled polymerization in undiluted fetal bovine serum (FBS) solution. RAFT polymerization initiated by glucose oxidase (GOx)−HRP enzymatic cascade catalysis was developed, opening up a new avenue to potential green synthesis of precision polymers by controlled radical polymerization in air. RDRP using an ATRP initiator was first introduced by Hawker and co-workers, capitalizing on the photoredox property of fac[Ir(ppy)3].16−19 Later, Boyer and co-workers developed visiblelight-controlled RAFT based on electron transfer from photocatalysts to RAFT agents (PET-RAFT).20,21 More recently, metal-free photocatalyzed RDRP has been developed for both RAFT and ATRP.22−25 Qiao and co-workers have studied the use of RAFT agents as the sole control agents without any external radical initiator or photocatalyst in visible light photopolymerization by activating the spin-forbidden n → π* transition of RAFT agents.26 The use of visible light to control RDRP and other polymerizations is extremely promising in developing sustainable chemistry by harnessing the abundant sunlight. Another promising direction for sustainable production of polymers is biocatalysis. Enzymatic biocatalysis is the basis of various in vivo biochemical reactions that provide the function of our daily life. Biocatalysis has also been widely utilized in organic synthesis and biotechnology that proceeds in a mild, efficient, and selective manner.27,28 Enzymes have been actively engaged in the polymer field. For example, a large array of polymers and polymer assemblies have been elaborated that
1. INTRODUCTION Reversible deactivation radical polymerization (RDRP) techniques are a powerful set of tools for the synthesis of well-defined polymers with controlled molecular weight, molecular weight distribution, composition, chain topology, and architecture.1−3 RDRP that has gained significant popularity includes nitroxidemediated polymerization (NMP),4 atom transfer radical polymerization (ATRP),5 single-electron transfer living radical polymerization (SET-LRP),6 and reversible addition−fragmentation chain transfer (RAFT) polymerization.7 RAFT polymerization is one of the most versatile synthetic techniques because of its applicability to most vinyl monomers, tolerance to functional groups, and compatibility with a wide range of conditions. Over the past years, significant advances have been made to address some of the limitations of RAFT,8 which have been designed to improve livingness,9 to overcome the necessity for oxygen-free conditions,10 to broaden the monomer class that RAFT agents can control,11 and to obtain ultrahigh molecular weight.12 As a result, the utility of RAFT has been considerably enhanced, greatly facilitating the synthesis of architectures with increasing complexity and the access to new materials. Recently, significant effort has been devoted to external regulation of RDRP.13 Photoregulated RDRP is particularly attractive because of temporal and spatial control as well as ready availability of light sources.14,15 Visible-light-induced © XXXX American Chemical Society
Received: August 27, 2015 Revised: October 7, 2015
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DOI: 10.1021/acs.macromol.5b01893 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Scheme 1. Proposed Mechanism of HRP-Initiated RAFT Polymerization
respond to enzymes.29−37 These enzyme-responsive materials are promising in detection, diagnosis, and drug delivery.38 Enzymes and proteins have also been the targets for bioconjugation with polymers.39−41 For this, ATRP, SETLRP, and RAFT that can be conducted under biologically relevant conditions are particularly useful.42−47 Transesterification catalyzed by lipase has been explored to in situ generate functional monomers for polymerization by RDRP.48−50 Polymer synthesis via enzymatic catalysis has been a longlasting goal in green and sustainable chemical processes.51,52 Well-established examples of synthetic polymers produced by enzymatic catalysis include degradable polyesters via ringopening polymerization and polycondensation catalyzed by lipase53 and conducting polyanilines via oxidative polymerization catalyzed by horseradish peroxidase (HRP).54 Despite a long history of biocatalysis in polymerization, employing enzymatic catalysis in RDRP with the aim to generate well-defined polymers emerged only recently. In 2011, di Lena and co-workers reported their study on enzymatic catalysis to control radical polymerization, whereby laccase/ HRP, ascorbic acid, and alkyl halides were used as the catalysts, reducing agent, and initiators, respectively.55,56 While a mild level of control was realized in the polymerization of poly(ethylene glycol) methyl ether acrylate (PEGA), with dispersities Đ (Mw/Mn) larger than 1.5 being obtained in most cases from medium to high conversions,56 the control was totally lost in the polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA), with gelation being observed within a short period of polymerization time.55 In the latter case, when the alkyl halide was replaced by a RAFT agent, no polymerization was observed, suggesting that this particular system does not catalyze RAFT. A certain level of control was possible only when both an alkyl halide and a RAFT agent were
present,55 for which the exact mechanism is still unclear. Independently, Bruns and co-workers reported similar results for the polymerization of N-isopropylacrylamide under ATRP conditions, which was catalyzed by HRP with sodium ascorbate as the reducing agent. The polymerization stopped at 48% conversion with dispersities larger than 1.44.57 Later, Bruns and co-workers investigated the use of hemoglobin and red blood cells to catalyze ATRP.58 While polymerization of N-isopropylacrylamide was not controlled, polymerization of PEGA and PEGMA showed a certain level of control, but the dispersities started to increase at moderate conversions. While these seminal works demonstrated the exciting potential for use of enzymes to catalyze ATRP, only limited success has been achieved and extension of enzymatic catalysis to other RDRP remains a challenge. A particular strength of RAFT, in comparison with other RDRP techniques, is that it enjoys similar conditions as in traditional radical polymerization with the only exception being that a RAFT agent is added to render polymerization under control by lowering the concentration of active radicals. Given the perceived value of enzymatic catalysis in traditional radical polymerization,59−67 we envisioned that enzyme-catalyzed, well-controlled RAFT polymerization should be feasible if the RAFT agent and the enzyme being used are mutually compatible and the enzyme retains its activity during polymerization. Herein, we report our study on HRP-initiated RAFT polymerization, which provided control for polymerization of a wide range of monomers (both more and less activated monomers) under a variety of conditions (solution polymerization, dispersion polymerization, and in biological milieu). Moreover, enzymatic cascade catalysis by GOx−HRP was investigated for RAFT polymerization in air. We are aware that Stevens and co-workers recently reported an elegant study B
DOI: 10.1021/acs.macromol.5b01893 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Scheme 2. Structures of (A) Monomers and (B) CTAs Used in This Study
of combining GOx with RAFT polymerization.68 However, in their study GOx was used only for deoxygenation by converting oxygen to hydrogen peroxide rather than catalyzing the RAFT process. To the best of our knowledge, this work represents the first report of enzyme-initiated RAFT polymerization that provides excellent control.
First of all, enzyme stability and activity are highly dependent on pH. Initiation efficiency of the ternary system is also affected by pH because it was proposed that the enol form of ACAC is the actual substrate that is responsible for radical generation,61 and higher pH promotes tautomerization toward enol against keto. Lastly, RAFT agents are susceptible to hydrolysis in aqueous solution and basic solution should be avoided.70 Therefore, we first investigated suitable pH for HRP-initiated RAFT polymerization of dimethylacrylamide (DMA) using chain transfer agent CTA1 (Scheme 2) in buffer solutions with pH values ranging from 3.5 to 7. The conversion of 2 h polymerization was used to evaluate the effect of pH (Table S1 and Figure S1). At pH 3.5, no conversion was observed. As the pH value increased from 4 to 7, conversion was observed to increase from 10% to 94%. Importantly, very narrow molecular weight distributions (Mw/Mn < 1.2) were observed for the polymerizations with high conversions (>80%). These results were encouraging in that both a high polymerization rate and a low dispersity were possible at pH close to neutral conditions, suggesting high enzymatic activity is attained under these conditions. These results are also consistent with the enol tautomer of ACAC being the substrate for radical generation.61 Taking into account RAFT agent hydrolysis stability, we decided to conduct systematic investigation of HRP-initiated RAFT polymerization at pH 7. To ascertain the indispensable role of each component of the HRP/H2O2/ACAC ternary initiating system, control experiments were conducted at pH 7 in the absence of one of the three components each time, and no detectable conversion was observed in each case, suggesting that RAFT polymerization was indeed initiated by this ternary system as a whole. Next, the versatility of this HRP-initiated RAFT polymerization was thoroughly investigated for both more and less activated monomers, in both homogeneous solution and heterogeneous dispersion polymerization as well as in biological solutions of fetal bovine serum (FBS). 2.2. HRP-Initiated RAFT Solution Polymerization of More Activated Monomers. DMA as a representative acrylamide monomer was first studied for HRP-initiated RAFT polymerization in phosphate buffer (pH 7), using CTA1 as a water-soluble RAFT agent at 30 °C. The monomer concentration was maintained at 10% w/v (approximately 1 M). The molar ratio of [DMA]:[CTA1]:[HRP]:[H2O2]: [ACAC] was maintained at 200:1:0.00054:0.2:4, unless otherwise stated.
2. RESULTS AND DISCUSSION 2.1. HRP-Catalyzed Initiation. HRP (E.C. 1.11.1.7) is a heme-containing metalloprotein that catalyzes oxidation of certain organic compounds such as phenols, anilines, and βdiketones using hydrogen peroxide or alkyl peroxides as the oxidants.69 The HRP/H2O2/acetylacetone (ACAC) ternary initiating system was developed to initiate traditional free radical polymerization of vinyl monomers almost two decades ago.59 It has generally been accepted that HRP catalyzes electron transfer by transforming a two-electron oxidation of ferriprotoporphyrin by H2O2 into two subsequent singleelectron transfer steps to generate two ACAC radicals, which then initiate polymerization of vinyl monomers.51,52 Although this enzyme-initiated radical polymerization has been well perceived as an environmentally benign and sustainable chemical process, it is surprising that this potential has not been explored in RDRP. The particular strength of RAFT being readily coupled with various radical generating processes makes it a great opportunity to develop a novel HRP-initiated living/ control radical polymerization. Hence, we rationalized that HRP-catalyzed generation of ACAC radicals by H2O2 would make the first step (step I in Scheme 1) in this enzymatic RAFT polymerization. Once ACAC radicals are generated, they participate in polymerization according to the well-established RAFT mechanism,3 as outlined in steps II−VI. As we can see, enzymatic action is confined to the first step, and HRP is not involved in subsequent RAFT steps. This simple but clear-cut feature makes this enzymatic RAFT distinct from previous studies on enzymatic ATRP where enzymes play roles in both activation of dormant chains and deactivation of propagating chains. We believe the essential deconvolution of the enzymatic initiation step from the rest of RAFT steps is exactly what makes enzymatic RAFT polymerization well-controlled as in a typical RAFT polymerization initiated, for example, by thermal initiators, which also vividly reflects the power of RAFT that is amenable to a wide range of initiating methods. In considering experimental conditions, we believed pH of the solution is of paramount importance for several reasons. C
DOI: 10.1021/acs.macromol.5b01893 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. Results of HRP-initiated RAFT polymerization of DMA under conditions [DMA]:[CTA1]:[HRP]:[H2O2]:[ACAC] = 200:1:0.00054:0.2:4, [DMA] = 10% w/v, buffer solution pH 7, 30 °C. (A) Pseudo-first-order kinetics, (B) dependence of molecular weight and dispersity on conversion, and (C) evolution of GPC traces over polymerization time.
Figure 2. 1H NMR spectrum and MALDI-ToF spectrum of PDMA32 synthesized by HRP-initiated RAFT polymerization using CTA1.
that HRP-initiated polymerization in the presence of a suitable CTA was well controlled, showing all the characteristics of a “living” RAFT polymerization under mild conditions. Because the HRP/H2O2/ACAC ternary initiating system is responsible for the generation of ACAC radicals that determine the polymerization rate, the concentration of the ternary initiating system was adjusted while keeping the three components at a constant molar ratio to see this effect. Thus, for a target number-average degree of polymerization (DP) of 200, the molar ratio of [CTA1]:[HRP]:[H2O2]:[ACAC] was changed from the initial 1:0.00054:0.2:4 to 1:0.000108:0.04:0.8, meaning that the concentration of the ternary initiating system
Polymerization of DAM initiated by HRP/H2O2/ACAC was very rapid, with more than 95% and near-quantitative conversion being reached within 1 and 2 h, respectively. Excellent linear kinetics, as expected for a pseudoliving polymerization, was observed for the semilogarithmic plot of monomer conversion vs polymerization time (Figure 1). Molecular weight of the produced PDMA also increased in a linear fashion with monomer conversion up to near-quantitative conversion. Dispersity Đ (Mw/Mn) gradually reduced as the polymerization proceeded and the final value of which was below 1.1, corroborating well with the GPC traces, which showed symmetric and unimodal peaks. These results indicate D
DOI: 10.1021/acs.macromol.5b01893 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules was reduced by a factor of 5. As expected, monomer conversion was lowered to 72% from 98% in 2 h, and a final conversion of 85% was obtained after 5 h. Again, the produced polymers using this lower concentration of ternary initiating system were unimodal with dispersities being lower than 1.1 (Figure S2). HRP acts on both H2O2 and ACAC as the substrates. While H2O2 was used as the limiting reagent with the molar ratio of [H2O2]:[CTA] being much smaller than 1 such that a low concentration of radicals can be maintained in order to achieve a high degree of livingness, cosubstrate ACAC was typically used in excess in order to accelerate the enzymatic catalysis according to the Michaelis−Menten kinetic model.71 A stoichiometric amount of ACAC ([CTA1]:[HRP]:[H2O2]: [ACAC] = 1:0.00054:0.2:0.4) was also tested to elucidate its effect on the polymerization rate. A mild reduction of monomer conversion from 98% to 90% within 2 h of polymerization was observed (Figure S3). These results suggest that both the concentration and molar ratio of the ternary initiating system can be manipulated to affect the polymerization rate. Structural analysis of a PDMA with a small DP was performed using 1H NMR, MALDI-ToF, and UV−vis to confirm that this HRP-initiated polymerization operates via RAFT mechanism. For this, a PDMA synthesis with a target DP of 50 was stopped at ∼63% conversion, resulting in a theoretical DPth of ∼32. The theoretical molecular weight (Mth = 50 × conv × MDMA + MCTA1 + MNa − MH, MDMA, MCTA1, MNa, and MH being the molar mass of DMA, CTA1, Na, and H, respectively) of this polymer was calculated to be 3457 g/mol. The polymer was thoroughly purified by extensive dialysis and isolated by freeze-drying for structural analysis. As shown in Figure 2A, the CH3− (a) and −CH2− (b) proton resonances derived from the Z-group of CTA1 appear at 1.2 and 3.3 ppm, respectively, suggesting the RAFT end groups were retained in the synthesized PDMA. Analysis of the proton resonances from the R-group was difficult due to their overlap with the major polymer proton resonances. This end-group analysis from 1H NMR provided a DPNMR of ∼33 and MNMR of 3556 g/mol. The major population in the MALDI-ToF spectrum (Figure 2B) is separated by 99 u, corresponding to the exact mass of one monomer unit. The observed mass (3455.64) agrees well with the calculated one (3455.23), as expected for a PDMA32 bearing both end groups derived from CTA1. Analysis of other minor populations in the MALDI-ToF spectrum suggested that these species also came from the expected RAFT-derived polymers (Figure S4). In addition, this PDMA polymer showed a strong absorption centered at 307 nm in the UV−vis spectrum (Figure S5), characteristic of trithiocarbonate RAFT agents. Taken together, these results unambiguously confirm that HPR-initiated polymerization of DMA in the presence of CTA1 was excellently controlled by the RAFT mechanism and a high degree of end-group fidelity was maintained. The high end-group fidelity of HRP-initiated RAFT polymers was further validated by and exploited in chain extension to prepare PDMA block homopolymers. For this, each block with a target DP of 200 was prepared without intermediate separation steps. Sequential polymerization afforded PDMA 176 (M n = 15.4 kg/mol, Đ = 1.11), PDMA176−PDMA211 (Mn = 38.3 kg/mol, Đ = 1.12), and PDMA176−PDMA211−PDMA209 (Mn = 59.0 kg/mol, Đ = 1.18) with monomer conversion being 88%, 94%, and 98% for the three steps, respectively. Successful block extension was evident from the GPC traces shown in Figure 3 as the molecular weight
Figure 3. GPC traces of PDMA polymers synthesized by sequential chain extension without intermediate purification.
clearly shifted to lower elution time and hence higher molecular weight. It is worth noting that the diblock and triblock PDMA homopolymers were prepared at very high conversions, but a low dispersity was still attained after three steps of synthesis. Next, PDMAs with a broad range of molecular weights, targeting DPs from 50 to 10 000, were prepared (Table 1). The Table 1. Summary of PDMAs Synthesized by HRP-Initiated RAFT Polymerizationa entry
[DMA]/ [CTA1]
time (h)
conv (%)
actual DP
Mth (kg/mol)
Mn (kg/mol)
Mw/Mn
1 2 3 4 5 6 7
50 100 200 500 1000 5000 10000
12 2 2 4 10 16 30
98 90 98 98 94 94 78
49 90 196 490 940 4700 7800
5.1 9.2 19.7 48.8 93.5 466.2 773.5
5.4 11.3 22.1 60.4 100.8 486.5 518.6
1.14 1.10 1.07 1.09 1.10 1.18 1.28
a
Conditions: [CTA1]:[HRP]:[ H2O2]:[ACAC] = 1:0.00054:0.2:4, [DMA] = 10% w/v, pH 7, 30 °C.
GPC traces in Figure 4A show that well-defined polymers with wide-ranging molecular weights were synthesized successfully. Molecular weight distribution was very narrow with Đ below 1.2 except for the PDMA targeting an ultrahigh molecular weight (106 g/mol) (Table 1, entry 7), which gave a reasonably low Đ of 1.28, albeit with a significant low-molecular-weight tailing. The solution became extremely viscous, and stirring was difficult at the late stage of synthesis for this polymer. The molecular weights of these polymers were close to theoretical ones and scaled linearly with their actual DPs, except for the one targeting a DP of 10 000 (Figure 4B). The significant departure of the observed molecular weight from its corresponding theoretical one in this case is ascribed to the significant tailing toward low molecular weight. We were aware that the polymerization rate was lower when targeting low DPs (50−100) than when targeting moderate DPs (200−500). While the exact reason was unknown at the present stage, we suspect that HRP activity might be inhibited at high CTA concentrations (low target DPs). Much longer polymerization time was needed when targeting exceptionally high DPs (5000−10 000) due to significantly reduced HPR concentrations at a constant reagent ratio and monomer concentration. Interestingly, for the polymerization with a target DP of 5000 (Table 1, entry 6), 92% conversion was reached within 6 h, although maintaining the polymerization for an additional 10 h E
DOI: 10.1021/acs.macromol.5b01893 Macromolecules XXXX, XXX, XXX−XXX
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Figure 5. GPC traces of (meth)acrylate polymers synthesized by HPR-initiated RAFT polymerization under conditions [M]:[CTA1]: [HRP]:[H2O2]:[ACAC] = 200:1:0.00054:0.2:4, [M] = 10% w/v, buffer solution pH 7, 30 °C.
Figure 4. PDMAs of different molecular weights synthesized by HRPinitiated RAFT polymerization: (A) GPC traces; (B) dependence of molecular weight on DP.
led to only a slight increase in conversion (94%); the GPC trace was still highly symmetric (Figure S6), suggesting that enzymatic catalysis was very mild such that keeping the polymerization even at high conversions for extended period of time does not have detrimental effect on the quality of resulting polymers. It is perhaps worth noting that the HRP concentration in the synthesis of PDMA with a target DP of 10 000 (Table 1, entry 7) was already as low as 5.4 × 10−8 M, but a reasonably high conversion of 78% was still achieved, demonstrating this enzymatic RAFT polymerization is extremely efficient. Having thoroughly studied HRP-initiated RAFT polymerization of DMA, we next extended the synthesis to other more activated monomer families, namely, acrylates and methacrylates. In each synthesis, sample withdrawing during polymerization was used to follow the polymerization kinetics and molecular parameters by 1H NMR and GPC (Figures S7−S10). As in the synthesis of DMA, polymerization of 2-hydroxyethyl acrylate (HEA), PEGA, and PEGMA all exhibited pseudo-firstorder kinetics, high monomer conversions (>88%) were achieved within 2 h, molecular weights followed a linear increase with conversion, and low dispersities (
