By far the most popular catalysts for the enzymatic initiation of radical polymerization (both of aromatic and vinyl monomers) are the so-called heme peroxidases. Particularly, the peroxidases from horseradish (HRP) and soybean (SBP) have been used most frequently.

These peroxidases contain protoporphyrin IX (heme, Figure 1) as prosthetic group with low-spin Fe^III in the resting state [16,17].

During the catalytic mechanism (Figure 2) the water ligand (intermediate 1) is substituted by hydrogen peroxide (or other organic hydroperoxides) resulting in a peroxo complex (intermediate 2). Upon heterolytic cleavage of the O-O bond the so-called compound I (Cpd-I) is formed. Cpd-I returns into the resting state by two individual hydrogen abstractions from the reducing substrates (In-H) resulting in the formation of two radical species (In·) which then initiate the polymerization process. Overall, peroxidases function as ‘electron relays’ coupling a two electron transfer step (reduction of peroxides to water or alcohols, respectively) to two subsequent single electron transfer steps.

H₂O₂ plays an ambivalent role in the peroxidase-initiated polymerization reaction. On the one hand, it serves as oxidant and therefore is essential for the catalytic action. On the other hand, if [H₂O₂] is too high, polymerization is inhibited twofold: (1) H₂O₂ is a known inactivator of heme [18,19]. It was proposed that in the presence of an excess of H₂O₂ Cpd-II reacts with another equivalent of H₂O₂ instead of returning to the resting state. The resulting Cpd-III decomposes along various pathways leading to irreversible enzyme inactivation [19]. (2) A catalase-like activity of peroxidases [20] at high [H₂O₂] results in the generation of O₂ thereby inhibiting the chemical polymerization reaction. This effect has been reported especially for the polymerization of vinyl monomers in form of a lag-phase. During this lag phase, remaining O₂ is consumed, probably by reaction with In. The resulting superoxide anion (O₂^·−) disproportionates quickly thereby overall accounting for a futile consumption of H₂O₂ and radical quenching. It should, however, be emphasized that the exact mechanism remains to be elucidated. Polymerization only proceeds if O₂ formed by the catalase-activity has been consumed [21].

The usual way of avoiding excess H₂O₂ is to add H₂O₂ in several portions, which turns out to be tedious and also bears the danger of leading to a certain degree of irreproducibility. Therefore, a range of in situ H₂O₂ generation methods have been proposed, [22,23,24] which might be useful also for peroxidase-initiated polymerizations. Indeed Uyama et al. and others reported a bienzymatic system for the polymerization of phenols comprising peroxidase as the initiation catalyst and glucose oxidase as in situ H₂O₂ generation catalyst (Figure 3) [25,26].

Interestingly, this system, despite its simplicity, has not found broad proliferation. Possibly, inhibitory effects of O₂ are quite severe in practice.

A rather unusual in situ generation of peroxo species was found accidentally by Samuelson and coworkers [27]. They found that a dioxane stabilizer (acetyl acetal) spontaneously hydrolyzed under the reaction conditions and was oxidized to peracetic acid, which then served as oxidant to initiate the polymerization of aniline (Figure 4).

Nevertheless, portionwise addition of H₂O₂ remains the most popular procedure to promote peroxidase-initiated polymerizations.

Laccases (E.C. 1.10.3.2) belong to the so-called blue-copper oxidases predominantly found in fungi but also in plants and insects [28,29,30,31]. Laccases catalyze hydrogen abstraction reactions from phenolic and related substrates resulting in corresponding phenoxy radicals. They contain four copper ions classified in one T1 copper ion and a T2/T3 cluster. It has been shown that the T1 site is the primary redox center accepting electrons from the electron donors. Thus, the fully oxidized laccase is transformed via four successive, fast single electron transfer (SET) steps into the fully reduced laccase. Molecular oxygen interacts with the fully reduced (T2/T3) cluster via a fast 2-electron-transfer process. The resulting peroxide is tightly bound so that release of H₂O₂ prior to the second 2-electron-transfer is efficiently prevented. As a result, the fully oxidized form of laccases comprising a μ₃-oxo-bridged trinuclear (T2/T3) site is formed. This structure is thermodynamically relatively stable and provides the driving force for the overall process and also provides an efficient electron transfer bridge to re-generate the fully reduced state (Figure 5).

Interestingly enough, the number of publications dealing with laccase-initiated polymerization falls back significantly behind peroxidase-initiated polymerization studies. This is astonishing insofar as the laccase-based systems appear to be significantly easier. One apparent advantage is that laccases utilize molecular oxygen instead of hydrogen peroxide as oxidant, but inhibition may prevent widespread use.

Often direct oxidation of the monomers is not possible due to unfavorable steric interaction with the enzymes’ active sites and/or due to unfavorable redox potentials. In such cases, initiation of the polymerization reaction may take place by so-called laccase- or peroxidase mediator systems (LMS or PMS, respectively). Here, enzymatic chain initiation does not proceed directly on a monomer molecule but indirectly via a small, oxidizable mediator (Figure 6). Two mechanisms can be distinguished: First, the mediator can serve as radical transfer catalyst without being incorporated into the final product. Phenothiazenes, for example, have been used to initiate the polymerization of sterically demanding phenols, [32,33] or ABTS to facilitate pyrrole polymerization [34,35]. Also transition metals (e.g., Mn^2+/3+) [36] or polyoxometallates [37] have been reported. Second, the mediator radical itself can initiate chain growth thereby being incorporated into the polymer (see Section 2.4). This mechanism is the predominant pathway for the polymerization of vinyl monomers with β-diketones representing the mediators of choice (vide infra) but may also be applicable for the polymerization of non-phenolic aromatics such as thiophenes [38].

Very recently the groups around Bruns [39] and di Lena [40,41] independently reported on biocatalytic atom transfer radical polymerizations.

Using laccases or heme-enzymes such as catalase of HRP, an ATRP-like polymerization was achieved with ascorbic acid as reductant and α-bromoesters as initiators. Despite the very early stage of development (particularly further mechanistic studies clarifying the initiation reaction are needed), this approach represents already now a very promising biocatalytic alternative to the existing toolbox [42]. Exciting further developments are expected here in the near future. Noteworthy, this approach also represents the first example(s) of a reductively initiated radical polymerization.

Ximenes and coworkers reported on the H₂O₂-independent Acac oxidation by HRP in the presence of molecular oxygen [43]. Intermediate formation of Acac^. and superoxide was postulated, which might potentially be exploited for the initiation of radical polymerizations. If feasible, such a pathway might open up new possibilities while circumventing the limitations of H₂O₂-promoted chain initiation reactions mentioned above. It remains to be shown whether such a mechanism might be fruitfully exploited for peroxidase-initiated polymerization reactions.

The major concern in vinyl polymerization is to control the polymer weight and polydispersity of the final product. In this respect, most studies have focused on the influence of biocatalyst-, mediator-, and oxidant concentration.

The biocatalyst concentration directly correlates with the chain initiation rate and thereby influences the number of growing chains in the reaction mixture and competing for the remaining monomers. As a rule of thumb, the higher the biocatalyst concentration the lower the average polymer weight, as demonstrated for peroxidases [44] and laccases [45]. At very high enzyme concentration, the initiation rate can be so dominant that only oligomers are formed. Under denaturizing conditions, too low enzyme concentrations manifest in low conversions due to enzyme inactivation [46].

Like enzyme concentration, choice and concentration of the mediator can have a major impact on the polymer properties. This was demonstrated by Kaplan and coworkers on the HRP-initiated polymerization of styrene and by Marechal and coworkers for the polymerization of acrylamide (Table 1) [47,48].

So far, detailed studies clarifying the influence of ß-diketone structure on enzyme activity are missing. Most likely, both steric and electronic effects influence the rate of the enzymatic initiation reaction as well as the rate of the chain growth (at least at an early stage of the chain growth).

It is generally assumed that the enol form represents the actual substrate for the enzymatic radical formation reaction (Figure 7) [49].

The state of the keto-enol equilibrium should determine the actual substrate concentration. Hence, high pH values should be beneficial as well as using elevated substrate concentrations. However, these measures may also be counteracted by a decreasing activity of the biocatalyst under these conditions. Principally, electron withdrawing substituents should increase the enzyme rate by shifting the keto-enol equilibrium and lowering the redox potential on the one hand. On the other hand, the resulting radical might be too stable due to very low lying SOMO energy.

Obviously, the ratio of monomer to initiator has a strong influence on the average size of the resulting polymer. The number of growing polymer chains, competing for the remaining monomer reservoir, increases with initial initiator concentration (Figure 8) [44].

There is an ongoing discussion about the necessity of ß-diketones to initiate the polymerization reaction. Earlier reports on ß-diketone-free polymerization of acrylamide [13,50] could not be reproduced by others [48,51]. One potential solution of this apparent discrepancy may be found in the very high H₂O₂ concentration in the mediator-free polymerizations resulting in ‘unusual’ heme-iron species [19]. Thus, highly reactive peroxidase-species might initiate the polymerization reaction by direct H-atom abstraction from the monomer. Alternatively, oxidative degradation of heme resulting of Fe-release into the reaction medium might account for catalytic, Fenton-like generation of reactive oxygen species (ROS) initiating the polymerization reaction.

Kobayashi and coworkers reported on the mediator-free direct polymerization of acryl amide using the laccase from Pycnoporus coccineus suggesting a simplified polymerization scheme [52]. Using the laccase from Myceliophthora thermophilia, this was not achieved [14,45] and a follow-up study validating Kobayashi’s initial study is still missing.

Particularly when using laccases, molecular oxygen plays an ambivalent role. On the one hand, O₂ serves as stoichiometric oxidant to initiate the polymerization reaction. On the other hand, excess O₂ can also efficiently quench the radical polymerization [45,53].

Recently, Nieto et al. reported on an interesting approach to balance the in situ O₂ concentration for laccase-initiated polymerization by co-application of glucose oxidase as additional O₂-consuming reaction [54].

In contrast to the above-mentioned polymerization of vinyl monomers, regioselectivity also plays an important role in the polymerization of phenols and anilines. Hence, many efforts have been directed towards gaining control over the chemo- and regioselectivity of the enzymatic polymerization of phenols and anilines.

Flavonoids represent a class of natural secondary metabolites that have found numerous applications in human health products. Especially their antioxidant and radical scavenging activity but also anti microbial properties make this product class interesting. The group around Kobayashi has investigated the production and properties of polymerized polyphenols [11]. Interestingly, in the majority of these examples, the antioxidant activity (and quite frequently also other health properties) are significantly increased as compared to the monomers. A unifying explanation for this observation is missing so far.

Early reports dealing with modified flavonids concern the direct laccase- and/or peroxidase-mediated polymerization of the monomers. More recently, oxidative conjugation to other polymers has gained considerable interest (Figure 18). Table 2 gives an overview over the polymeric products reported.

As mentioned above, the oxidative grafting of flavonoids to polymers is enjoying increased attention especially aiming at functionalized textiles or food applications [130].

One of the first examples for this was reported by Kobayashi and coworkers who oxidatively grafted poly(allylamine) with catechin (Figure 19) [131], which was followed up more recently by Rao and coworkers [132,133].

Instead of poly(allylamine) also natural polymeric support can be used resulting in a composite with similarly superior antioxidant activity as compared to the catechin monomer. For example gelatin was grafted with catechin [134]. Wool can also be grafted e.g., with gallates resulting in composites with antioxidant and antimicrobial activity [135]. When using gallic acid esters, increased water repellence was achieved. Chitosan-composites with gallic acid (derivates) [136,137,138,139], ferulic acid [140], flavonoids [141] exhibit increased antioxidant activity. An interesting extension of this concept is to use amine-functionalized inorganic supports [142].

A practical application of this grafting technology may be in the textile and food industry yielding products with antimicrobial activity and modified rheological properties [130,138,139,143,144,145,146].

Oxidoreductase-initiated covalent linkage of phenols has also received considerable interest for the cross-linking of polymers to form biocompatible hydrogels. For example, proteins presenting tyrosine moieties at their surface can be gelled in the presence of laccases or peroxidases/H₂O₂ (Figure 20) [147,148,149,150].

Next to proteins also natural polymers exhibiting phenolic side chains can be gelled enzymatically. For example pectins contain some ferulic acid which can be used for oxidative crosslinking of the pectin chains for irreversible gellation [151,152,153]. However, even if no phenolic side chain is present in the polymer, it may be introduced chemically. In that respect, tyramine has gained some popularity e.g., for the crosslinking of carboxymethyl cellulose [154], alginate [155], hyaluronic acid [156], dextran [157] or artificial polymers such as Tetronic [158].

NH₂-containing polymers (e.g., chitosan) can be modified with phenolic carboxylic acids via amidation [159] or directly act in Michael-fashion on enzyme-generated quinones (Figure 21) [146,160]. For example, tyrosinases generate reactive quinones from phenols, which can react with chitosan [141,145,160,161]. Similarly, reactive quinones can be generated using laccases [140,146] of peroxidases [136,139].

A very creative application of the system HRP/H₂O₂/Acac for graft polymerization of acryl amide on starch was reported recently by Biswas and coworkers [114]. A biocatalytic alternative route to the established cerium(IV)-based routes to poly(acrylate)-grafted starch with applications as superabsorbersor performance additives in paper making or textile sizing was reported. Under non-optimized conditions grafting efficiencies of up to 65% are reported. The authors suggest covalent attachment of the poly(acrylamide) chain to the starch backbone via H-abstraction at the glycosidic C-atom (Figure 22).

Poly(acrylamide) grafting onto lignin has been reported in the presence of organic hydroperoxides [162,163]. The proposed mechanism (Figure 23.) comprises laccase-catalyzed H-atom abstraction from phenolic lignin residues. The resulting phenoxy radicals interact with the peroxides generation alkoxy- and hydroperoxy-radicals, which are supposed to initiate the acrylamide polymerization process. Grafting of poly(acrylamide) onto lignin is proposed to occur through recombination of the respective radicals.

Urushiols are a class of natural alkyl-substituted phenols and catechols (Figure 24), which in the presence of laccase can polymerize to form Urushi, a natural lacquer used e.g., in traditional Japanese artwork.

Due to its potential as ‘fully renewable and benign’ alternative to chemical formaldehyde-phenol resins, the enzymatic polymerization of urushiols has been thoroughly investigated by the Kobayashi group [164,165,166,167].

Synthetic, tailored urushiols have also been proposed by lipase-catalyzed acylation of benzylic OH-groups followed by peroxidase- or laccase-initiated polymerization of the phenol/catechols moiety (Figure 25).

Cardanols are natural phenols structurally related to urusiols and likewise can be polymerized using laccases of peroxidases [32,33,168,169,170].

One advantage of oxidoreductase-catalysis that is frequently mentioned is its high selectivity. As mentioned above, so far no examples of stereospecificity have been reported in enzyme initiated polymerization, and are not very likely to come up in the near future. However, there is a range of examples demonstrating chemoselective radical formation, which will be shortly outlined below.

The selective polymerization of bifunctional monomers containing vinyl and phenol moieties. Generally, laccases and peroxidases act on the phenol part selectively leading to vinyl-substituted poly(phenols) which then are available for further modification (Figure 26) [68,101,171].

A very nice example for the potential of biocatalysis as complementary tool to established chemocatalysis was reported using m-ethinylphenol as monomer [106]. Subjecting this compound to HRP/H₂O₂, poly(phenol) formation was the only reaction observed wile leaving the ethinyl group untouched. In contrast, Cu^I catalysts lead to dimerization via the ethinyl function (Figure 27).

Another example for chemoselectivity of enzymatic polymerization was reported by Bilici et al. [172]. Here, an aldehyde-bearing catechols was polymerized enzymatically without oxidizing the aldehyde moiety.

A good portion of the laccase- and peroxidase literature deals with bioremediation and pollutant control [130,173,174,175]. The removal of phenolics is rather straightforward as these compounds are generally oxidized by the laccases or peroxidases directly [176]. In most of the cases, the detoxification mechanism is based on the formation of insoluble oligomeric or polymeric products which can then be removed by filtration.

Hence, various cresols and derivates [177] can be oxidatively polymerized and thereby reduced in toxicity. Likewise halophenols [178,179,180,181,182] bisphenol A, [183,184,185] various hormones and so-called endocrine disrupting compounds [186,187,188] or even TNT [189,190] have been reported. Non-phenolic compounds may be ‘inactivated’ using the LMS or PMS strategy [36,191,192,193,194,195,196,197,198].