One-Enzyme Triple Catalysis: Employing the Promiscuity of

Dec 12, 2017 - We demonstrate a new concept in polymer chemistry that the promiscuity of enzymes, as represented by horseradish peroxidase, can be emp...
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Letter Cite This: ACS Macro Lett. 2018, 7, 1−6

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One-Enzyme Triple Catalysis: Employing the Promiscuity of Horseradish Peroxidase for Synthesis and Functionalization of WellDefined Polymers Zhifen Liu, Yue Lv, Anqi Zhu, and Zesheng An* Institute of Nanochemistry and Nanobiology, College of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China S Supporting Information *

ABSTRACT: We demonstrate a new concept in polymer chemistry that the promiscuity of enzymes, as represented by horseradish peroxidase, can be employed for RAFT polymerization and thiol−ene and Diels−Alder reactions to synthesize well-defined functional polymers, via three different catalytic reactions mediated by one single enzyme.

P

enzyme. The concept of employing the promiscuity of enzymes for efficient and versatile synthesis and functionalization of welldefined polymers may open up a new field in polymer chemistry. To demonstrate this new concept, herein we report HRP-initiated RAFT polymerization and postpolymerization functionalization by HRP-catalyzed thiol−ene26−28 and Diels− Alder29,30 reactions. While these and other “click” reactions have been widely adopted in polymer synthesis and functionalization,31−51 their enzyme-catalyzed processes have not been studied for functionalization of well-defined polymers. The demonstration of one enzyme for three different reactions is a novel concept, and the processes reported in this work may be well adapted to other systems. The potential impact that these “bioclick” reactions would have is particularly clear by analogy to well-recognized click reactions. Their use in the polymer field has created enormous possibilities for generating new materials and enabling new applications. We thus believe enzyme catalysis coupled with modern synthetic polymer chemistry is of significant interest. HRP catalyzes oxidation of acetylacetone (ACAC) with H2O2 to generate ACAC radicals, which subsequently initiate RAFT polymerization in the presence of suitable chain transfer agents (CTAs), in buffered aqueous solution (pH 7), at 30 °C.15,52 As shown in Scheme 1, this HRP-initiated RAFT polymerization was first employed to synthesize a copolymer of N,N-dimethylacrylamide and allyl acrylamide P(DMA97-coALAM11) P1 (Mn,GPC = 15.3 kg/mol, Đ = 1.29) and poly[poly(ethylene glycol) methacrylate] PPEGMA58 P2

olymer synthesis via enzymatic catalysis is well recognized as a sustainable strategy for preparation of polymer materials because of the mildness, efficiency, environmental friendliness, and bioavailability of enzymes.1 Given that various methods for enzyme stabilization and immobilization have been substantially developed, with some of them having already been commercialized (e.g., immobilized lipase by Novozyme),2−4 applying enzymes for polymer synthesis will continue to rise in the future. Traditionally, enzymes have been used for synthesis of polymers with wide molecular weight distributions,5−11 and employing enzymes for synthesis of precision polymers with predetermined molecular weights and narrow molecular weight distributions has only been a recent advent.12−17 Although it is generally perceived that enzymes are highly specific to the substrates they act on, we have noticed that horseradish peroxidase (HRP) shows a surprisingly high degree of promiscuity from our own study as well as from literature reports. It is well-known that HRP catalyzes oxidation of electron-rich aromatic compounds including phenol and aniline derivatives by peroxides to generate aromatic polymers.18−20 Perhaps, less known is that HRP also uses thiols as substrates to generate thiyl radicals or disulfides;21,22 HRP-catalyzed formation of thiyl radicals has been studied by Scott and coworkers23 for thiol−ene polymerization, while HRP-catalyzed disulfide formation has been used to construct degradable hydrogels and nanogels by Groll et al.24 More recently, Fox and co-workers discovered that aerial oxidation of dihydrotetrazine can be efficiently catalyzed by HRP to generate tetrazine for turning on Diels−Alder reaction with trans-cyclooctene.25 Collectively these studies convincingly point to a very interesting phenomenon: HRP indeed acts on a breadth of substrates in diverse reactions that may be potentially harnessed in synthesis of functional polymers based on just one single © XXXX American Chemical Society

Received: December 6, 2017 Accepted: December 8, 2017

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DOI: 10.1021/acsmacrolett.7b00950 ACS Macro Lett. 2018, 7, 1−6

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ACS Macro Letters

Scheme 1. HRP-Initiated RAFT Polymerization and Subsequent End-Group Removal (A), HRP-Catalyzed Thiol−Ene Functionalization of P3 (B), and Functionalization of P4 via Sequential Esterification and HRP-Catalyzed Thiol−Ene or Diels− Alder Reactions (C)

Figure 1. (A) 1H NMR spectra of P1, P3, and P5 in D2O (the box indicates changes of vinyl group); (B) GPC traces of P1, P3, and P5; (C) FTIR spectra of P3 and P5 (the blue line indicates the disappearance of a vinyl group).

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DOI: 10.1021/acsmacrolett.7b00950 ACS Macro Lett. 2018, 7, 1−6

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ACS Macro Letters

Figure 2. (A) 1H NMR spectra of P2, P4, P6, and P7 in DMSO-d6 (the box indicates changes of vinyl group, and the arrows indicate the appearance of new methylene groups due to a thiol-end adduct); (B) GPC traces of P2, P4, P6, and P7; and (C) FTIR spectra of P6 and P7 (the blue line indicates the disappearance of the vinyl group).

Figure 3. (A) 1H NMR spectra of P2, P4, P8, and P9 in DMSO-d6 (the box indicates changes of the norbornene group, and the arrows indicate the appearance of new signals due to Diels−Alder adduct); (B) GPC traces of P2, P4, and P8; and (C) FTIR spectra of P8 and P9 (the blue line indicates the disappearance of the norbornene group).

(Mn,GPC = 29.2 kg/mol, Đ = 1.37). The relatively large dispersity of the latter polymer is probably caused by the crosslinker impurity in the monomer. As suggested by one of the reviewers, we also synthesized low-dispersity polyacrylamide copolymers with hydroxyl groups (see Supporting Information). The synthesis of copolymer P1 having pendant allyl groups, designed for thiol−ene reaction, was stopped at an intermediate conversion (∼55%) in order to minimize potential copolymerization of the allyl group with acrylamide and other side reactions involving the allyl groups. P2 was chosen for its excellent water solubility, and its hydroxyl groups can be converted to allyl- or norbornene-functionalized copolymers via esterification, which can then be used for subsequent modifications via thiol−ene or Diels−Alder reactions. The composition and number-average degree of polymerization were assigned via 1H NMR spectroscopy analysis (see Figures 1 and 2 and S2 and S3). To avoid potential interference of the trithiocarbonate end group on subsequent modifications, P1 and P2 were subjected to aminolysis and in situ thia-Michael protection of the generated polymeric thiols using excess ethanolamine and 2hydroxyethyl acrylate or DMA,53,54 to provide the corresponding P3 and P4, respectively. Both GPC and 1H NMR spectroscopy of P3 and P4 showed minimal changes in comparison with those for P1 and P2, as expected for removing a small group from a polymer chain end. Complete removal of the trithiocarbonate end group was confirmed by UV−vis photospectroscopy (Figures S4 and S5).

The hydroxyl groups of P4 were partially esterified with either 3-butenoic acid or 5-norbornene-2-carboxylic acid to afford vinyl- (P6) or norbornene-functionalized (P8) copolymer. Formation of ester was confirmed by 1H NMR and FTIR spectroscopies (Figures 2 and 3). Alkene proton resonances appeared at 5−6 ppm for P6 and 5.8−6.3 ppm for P8, and the intensity of the hydroxyl group resonances at 4.4−4.9 ppm was reduced. The degree of esterification was calculated to be 39%. The GPC trace of P6 (Mn,GPC = 30 kg/mol, Đ = 1.39) remained essentially unchanged, indicating a relatively neat esterification process, while the GPC trace of P8 (Mn,GPC = 33 kg/mol, Đ = 1.59) showed a high-molecular-weight shoulder, indicating a certain degree of ring-opening reaction due to the high reactivity of the strained norbornene group. In principle, this side reaction will result in an overestimate of the reaction yield for the Diels−Alder reaction. However, this effect is negligible since an excess dihydrotetrazine derivative was used in HRP-catalyzed Diels−Alder reaction (vide infra). The conditions for HRP-catalyzed thiol−ene reaction were first optimized using either oxygen or H2O2 as the oxidant. Thiol−ene modification of P3 using stoichiometric 2mercaptoethanol as a representative water-soluble thiol was first investigated for this purpose, and the results are summarized in Table S1. The catalytic role of HRP was confirmed by control experiments conducted without HRP, which practically did not proceed when H2O2 was used as the oxidant. Notably, high conversions were achieved in 1 h for HRP-catalyzed thiol−ene reactions in the presence of H2O2, 3

DOI: 10.1021/acsmacrolett.7b00950 ACS Macro Lett. 2018, 7, 1−6

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ACS Macro Letters Table 1. Summary of Diels−Alder Reactionsa

85% conversion for the reaction exposed to air, and quantitative conversion for the reaction under nitrogen protection. Thus, effective thiol−ene reactions require the simultaneous use of HRP and H2O2. The reaction yield was shown to be dependent on the concentration of the HRP/H2O2 catalytic system, as expected. On the other hand, HRP-catalyzed thiol−ene reaction using oxygen as the oxidant via direct exposure to air only gave a relatively low yield (20%), possibly due to the complex role of oxygen which can oxidize thiol to thiyl radical and further to disulfide or interfere with radical addition to alkene. Thus, HRP-catalyzed thiol−ene modification of P6 was conducted using H2O2 under protection of nitrogen, and quantitative conversion was again observed within 1 h with stoichiometric thiol. Quantitative thiol−ene reactions under optimized conditions were supported by the disappearance of the vinylic groups (5−6 ppm) in the 1H NMR spectra and the appearance of new methylene proton resonances: 2.6 ppm for P5 and 1.8 and 2.4−2.6 ppm for P7. The alkene vibrations,55 924 cm−1 (out-of-plane C−H bending) for P3 and 721 cm−1 (methylene rocking) for P6, also disappeared in the FTIR spectra. Furthermore, GPC analysis indicated that P5 (Mn,GPC = 15.7 kg/mol, Đ = 1.30) and P7 (Mn,GPC = 30 kg/mol, Đ = 1.39) had similar macromolecular characteristics as their precursor P3 and P6 due to the relatively small changes in molecular weight, from which well-controlled thiol−ene processes with minimal side reactions can be inferred. Previous work has shown that HRP can efficiently catalyze the oxidation of dihydrotetrazine derivatives to tetrazine derivatives, thus turning on the Diels−Alder reaction between tetrazine derivatives and trans-cyclooctene.25 Inspired by this progress, we were interested in applying this HRP-catalyzed Diels−Alder reaction for modification of polymers having more readily available norbornene group (e.g., P8). For this, an aqueous solution containing (4-oxo-4-(6-(6-(pyridin-2-yl)-1,4dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino) butanoic acid (DHTz-acid) and HRP was stirred at room temperature for 30 min in air to provide the corresponding tetrazine derivative (Tz-acid).25 Then P8 was added to the solution to start Diels− Alder reaction (norbornene/tetrazine = 1:2). Successful HRPcatalyzed oxidation of DHTz-acid to Tz-acid was confirmed by UV−vis photospectroscopy: a complete shift of the absorption peak from 290 to 325 nm and the appearance of a new peak at 525 nm (Figure S11). Diels−Alder reaction was complete within 1 h, as evidenced by 1H NMR and FTIR analysis (Figure 3). In the 1H NMR spectra, the unsaturated double bond (5.8− 6.3 ppm) and saturated methine protons (2.7−3.2 ppm) of the norbornene group in P8 disappeared or shifted after Diels− Alder reaction. Concomitantly, the proton resonances corresponding to the added norbornene moiety (2.3−2.6 ppm and 7−9.5 ppm) appeared in P9. In the FTIR spectra, the norbornene double bond (713 cm−1, alkene rocking) of P8 disappeared, and the characteristic vibrations of the added aromatic moiety of P9 appeared at 1600−1300 cm−1 (alkene stretching), providing further evidence for the success of the Diels−Alder reaction. However, even in the absence of HPR oxidation of DHTz-acid by oxygen, subsequent Diels−Alder reaction could still occur. We reasoned that oxidation of DHTz is pH-dependent as it would be increasingly difficult to oxidize the corresponding protonated species. By lowering pH, air oxidation and thus Diels−Alder reaction without HRP were significantly suppressed. For example, at pH 5, no reaction proceeded without HRP, but HRP-catalyzed reaction gave a mild yield (21%) after 12 h (see Table 1).

entry

P14-ene/DHTz-acidb

HRP (U/L)

pH

time (h)

conv. (%)

1 2 3 4c 5c 6 7 8 9 10

1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

2.95 2.95 -2.95 -2.95 -2.95 2.95 --

7 7 7 7 7 6.5 6.5 6 5 5

1 1 1 1 1 2 2 12 12 12

81 >99 41 7 -96 37 87 21 --

a

Reaction conditions: room temperature, open to air, 500 rpm, [P14ene] = 0.8 mM. bMolar ratio of norbornene moieties of P14/DHTzacid. cReactions conducted under nitrogen protection. Note: refer to Supporting Information for the synthesis and characterization of P14.

In conclusion, we have demonstrated a new concept in polymer chemistry employing the promiscuity of HRP for initiation of RAFT polymerization, thiol−ene, and Diels−Alder reactions. These three different catalytic reactions proceed efficiently under mild conditions, leading to the preparation of well-defined polymers and quantitative functionalization via a postpolymerization approach. The versatility of using renewable biocatalysts for various reactions useful for polymer synthesis and modification may open up an exciting new direction in the field of polymer chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00950. Experimental details and supplementary data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zesheng An: 0000-0002-2064-4132 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank financial support by National Natural Science Foundation of China (21674059) and assistance of Instrumental Analysis and Research Center (Shanghai University).

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