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ENZYMATIC POLYMERIZATION Introduction Enzymes catalyze not only all in vivo biosynthetic reactions in living cells for maintaining “life” but also many in vitro reactions of natural and unnatural substrates under selected reaction conditions. Enzymatic catalysis for organic synthesis possesses advantages such as much acceleration of reaction rate, operation under mild conditions, and high stereo-, regio-, and chemoselectivities of reactions in comparison with those of chemical catalysts. Such characteristic properties have brought about an extraordinarily rapid increase in interest in the area of biotransformations (1–5). All naturally occurring polymers are produced in vivo by enzymatic catalysis. Recently, in vitro synthesis of polymers through enzymatic catalysis (“enzymatic polymerization”) has been extensively studied (6–14); highly selective polymerizations catalyzed by enzymes have been developed to produce various functional polymers in response to increasing demands of structural variation of synthetic targets for polymers in material science. This article deals with recent advances in enzymatic polymerizations. We define enzymatic polymerization as “chemical polymer synthesis in vitro (in test tubes) via nonbiosynthetic (nonmetabolic) pathways catalyzed by an isolated enzyme.” Enzymes are generally classified into six groups. Table 1 shows typical polymers produced with catalysis by respective enzymes. The target macromolecules for the enzymatic polymerization have been polysaccharides, poly(amino acid)s, polyesters, polycarbonates, polyaromatics, vinyl polymers, etc. Here, enzymatic polymerizations are described according to the polymer structure. In many cases, enzymatic polymerization enables the synthesis of polymers, which otherwise are difficult to prepare. Enzymatic polymerization often provides an environmentally Encyclopedia of Polymer Science and Technology. Copyright John Wiley & Sons, Inc. All rights reserved.
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Table 1. Classification of Enzymes and In Vitro Production of Typical Polymers Catalyzed by Respective Enzymes Enzymes Oxidoreductases Transferases Hydrolases
Typical polymers Polyphenols, polyanilines, vinyl polymers Polysaccharides, cyclic oligosaccharides, polyesters Polysaccharides, poly(amino acid)s, polyamides, polyesters, polycarbonates
Lyases Isomerases Ligases
benign process, where starting materials and products are within the natural material cycle; this is in the context of “green polymer chemistry” (13,14).
Polysaccharides Polysaccharides are among the most important biopolymers as are proteins and nucleic acids in nature. They are regarded as three important families of natural biomacromolecules. As to the enzymatic polymerization for polysaccharides, hydrolases and transferases are reported to catalyze their synthetic reactions. Hydrolases. It is generally accepted that an enzymatic reaction is virtually reversible, and hence, the equilibrium can be controlled by selecting the reaction conditions. Based on this view, hydrolases, enzymes catalyzing a bondcleavage reaction by hydrolysis, have been developed as catalyst for the reverse reaction of hydrolysis, leading to polymer production by a bond-forming reaction (9,12). It is believed that using a glycosidase for the glycosylation process is one of the most promising methodologies for selective construction of a glycosidic linkage under appropriate conditions, since chemical approach requires complicated procedures including a regioselective blocking and deblocking of a hydroxy group in the sugar moiety to achieve regioselectivity, and furthermore, complete stereocontrol of the glycoside bond-formation has not often been achieved by chemical catalysts. Enzymatic formation of a glycosidic bond is realized by combined use of a glycosyl donor and a glycosyl acceptor. The former is to be activated by an enzyme to give a glycosyl-enzyme intermediate which can be attacked by a hydroxy group of the acceptor, forming a glycosidic bond between the donor and the acceptor. The repeated glycosylations are expected to produce polysaccharide molecules. Glycosyl fluorides, sugar derivatives whose anomeric hydroxy group is replaced by a fluorine atom, are known to be recognized by glycosidases. Cellulose is one of the most important biomacromolecules, which is the most abundant organic substance on the earth (12). Thus, in 1991, the first in vitro synthesis of cellulose via nonbiosynthetic pathway has been achieved by an enzymatic polymerization of β-cellobiosyl fluoride as substrate for Tricoderma viride cellulase, an extracellular hydrolytic enzyme of cellulose (Fig. 1) (15–21). The polymerization was performed in an aqueous organic solvent in order to make the desired
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Fig. 1. In vitro synthesis of artificial cellulose via cellulase catalysis.
polycondensation predominant in comparison with the competitive hydrolysis reaction. A mixed solvent of acetonitrile/acetate buffer (pH 5) (5:1) gave the best results in terms of the yield of water-insoluble “artificial cellulose.” The enzyme promoted transglycosylation of the cellobiosyl moiety toward the 4 -hydroxy group of another monomer eliminating hydrogen fluoride. In this polymerization, regioand stereoselectivities were perfectly controlled. The cellulase-catalyzed polycondensation of new cellobiosyl fluoride derivatives, 6-O-methyl and 6 -O-methyl-β-cellobiosyl fluorides, have been examined (22,23). The 6-O-methylated monomer was polymerized using the purified enzyme in a regio- and stereoselective manner to give a novel cellulose derivative having a methyl group alternatingly at the 6-position, which can never be realized by the conventional modification of natural cellulose, ie, methylation of cellulose. On the other hand, the 6 -O-methylated monomer gave a mixture of low molecular weight oligomers. The difference of the polymerization behavior can be explained by the steric repulsion between the methyl group of the monomers and the active site of the cellulase catalyst. The process of the artificial cellulose was visually analyzed by using transmission electron microscopy (24). Cellulose formation was detected as early as 30 s after the initial stage of the reaction in the aqueous acetonitrile. The electron diffraction pattern of the product showed the typical pattern of the crystal structure of thermodynamically stable cellulose II with antiparallel orientation between each glucan chains. When the purified cellulase (39 kDa) was used, cellulose microfibrils with an electron diffraction pattern characteristic of metastable cellulose I with parallel orientation, an allomorph of natural cellulose, were first observed in an artificial process (25). Based on these results, a new concept of choroselectivity, selectivity concerning the relative ordering of the polymer chain direction, in polymerization chemistry has been proposed (26–28). In some cases, the enzymatic polymerization afforded spherulites of artificial cellulose II, composed of single crystals with the molecular axis orientated perpendicular to the plane (29). Both positive- and negative-type spherulites were observed by polarization optical microscopy. By changing the reaction parameters,
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the size, growth and formation/degradation rate, and number of spherulites could be controlled. α-Amylase catalyzed the polycondensation of α-D-glucosyl fluoride in an aqueous solution to produce maltooligosaccharides (mainly pentamer) (30). αD-Maltosyl fluoride was also polymerized by α-amylase catalyst in an aqueous methanol, yielding a maltooligosaccharide with degree of polymerization (DP) up to 7 (31). Enzymatic transglycosylation of α-D-maltosyl fluoride with a cyclodextrin using pullulanase or isoamylase as a catalyst produced a branched cyclodextrin, 6-O-α-maltosylcyclodextrin (32,33). Synthetic xylan was synthesized by a cellulase-catalyzed polymerization using β-xylobiosyl fluoride as a substrate (34). The enzymatic polymerization proceeded in a perfect regio- and stereoselective manner to produce powdery artificial xylan, which is insoluble in any organic solvent. Xylan, one of the most important components of hemicellulose in plant cell walls, normally contains 4O-methylglucuronic acid or L-arabinose as a minor unit in the side chain. On the other hand, the artificial xylan consists exclusively of a xylopyranose moiety connected through a β(1→4) glycosidic bond. The first synthesis of a cellulose–xylan hybrid polymer, a novel polysaccharide having a glucose–xylose repeating unit, has been achieved by the xylanasecatalyzed polymerization of β-xylopyranosyl-glucopyranosyl fluoride (Fig. 2) (35, 36). Identification of the enzyme fraction promoting the polymerization showed that endoxylanase was highly efficient for production of the hybrid polymer. Cellulase-catalyzed polycondensation of 4-thio-β-cellobiosyl fluoride produced hemithiocellodextrins having 4-thiocellobiosyl repeating units linked by β(1→4) oxygen linkages (37). A water-soluble oligomer with DP up to 20 was obtained in an aqueous acetonitrile. Chitin is the most abundant organic macromolecules in the animal field found in invertebrates (12). The in vitro synthesis of this important biomacromolecule has been achieved for the first time by enzymatic ring-opening polyaddition of a chitobiose oxazoline monomer (Fig. 3). Chitinase, a hydrolysis enzyme of chitin, regio- and stereoselectively induced the polymerization of the monomer in a basic buffer (38–41). It is postulated that the monomer is preferable as a
Fig. 2. Enzymatic synthesis of cellulose–xylan hybrid polymer.
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Fig. 3. In vitro synthesis of artificial chitin via chitinase catalysis.
substrate because it can be recognized by the active site of chitinase readily due to the oxazoline structure resembling that of the transition state of the chitin hydrolysis with chitinase as revealed by a later work (12,42). Thus, the monomer is regarded as a “transition state analogue substrate” for chitinase. From x-ray diffraction and nmr analysis, the product was found to show crystal structure of α-chitin. A oxazoline monomer from N-acetyl glucosamine was also polymerized at high substrate concentration to give chitooligosaccharides. The visualization of high ordered structure formation during the enzymatic synthesis of artificial chitin has been investigated (43). Plate-like single crystals of α-chitin were first formed and gradually shaped into ribbons by the rapid growth along the a axis with the crystalline thickness being ca 10 nm. The α-chitin ribbons then aggregated to form bundle-like or dendritic assemblies as the ribbon concentration in solution increased. They grew up to spherulites by splaying and branching. This artificial chitin spherulite, in which a number of α-chitin ribbons radiated from a common center, is completely different from the helicoidal textures composed of α-chitin microfibrils known as a typical three-dimensional organization of chitin (see CHITIN and CHITOSAN). A cellulose–chitin hybrid polymer, a nonnatural polysaccharide having a glucose unit and an N-acetyl glucosamine unit alternatingly in the main chain, was synthesized by chitinase-catalyzed polyaddition of a disaccharide oxazoline monomer in an aqueous solution (44). Sugar-chain elongation from di-N-acetylchitobiose as initial substrate to hexamer and heptamer of chitooligosaccharide was efficiently induced through lysozyme catalysis in an acetate buffer containing 30% ammonium sulfate at 70◦ C. The high concentration of ammonium sulfate resulted in a remarkable increase of the hexamer and heptamer productions. In this reaction, a sugar-elongation from the dimer to trimer was the rate-limiting step in the overall process of transglycosylation (45). Transferases. Phosphorylase catalyzes polymerization of α-D-glucose-1phosphate in the presence of primer, leading to in vitro synthesis of amylose (Fig. 4) (46). By utilizing phosphorylase catalysis, various amylose derivatives such as linear-, star-, and comb-shaped amylose polymers were synthesized (47). The chain length could be controlled by a simultaneous start for all chains using a primer with a minimum length of four glucosyl residues. This method was
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Fig. 4. Phosphorylase-catalyzed synthesis of amylose.
applied to production of styryl-type amylose macromonomer (48), amylose-graftpoly(dimethylsiloxane) (49), amylose-graft-poly(L-glutamic acid) (50), amyloseblock-polystyrene (51), amylose-block-poly(ethylene oxide) (52), and amylosecontaining silica gel (53,54). Amylose-polytetrahydrofuran (PTHF) inclusion complex was synthesized by the phosphorylse-catalyzed polymerization in the presence of PTHF (55). Cyclodextrin-α(1→4)glucosyltransferase (CGTase) catalyzes formation of cyclodextrins from starch. By use of immobilized CGTase (silica gel support functionalized with glutardialdehyde), α-glucosyl fluoride was transformed in high yields, predominantly into cyclodextrin and maltooligomers as by-products (56). α-Maltosyl fluorides substituted at the 6- or 6 -position with H, F, Br, OCH3 , and OCOCH3 have been tested as substrates for CGTase (57). Among these substrates, only 6 -O-CH3 and 6 -O-COCH3 monomers were polymerized to give the cyclic compounds, indicating that the affinity of substrates toward the catalytic site of CGTase (Bacillus marcerans) greatly affected the specificity of the cyclization.
Poly(amino acid)s Biosynthesis of artificial polypeptides has been achieved by the expression of target proteins in living cells with a gene recombination technique; polypeptides with precise control of the chain length, sequence, and stereochemistry have been synthesized by genetic engineering. On the other hand, it is well known that amino acid derivatives are subjected to protease-catalyzed coupling reaction, yielding functional peptide compounds (58). In using amino acid esters as monomer, poly(amino acid)s are obtained. Papain catalyzed the polymerization of L-methionine methyl ester hydrochloride to give water-insoluble oligomer with DP = 8–10 (59–61). The resulting water-insoluble oligomer was converted to water-soluble sulfoxide and sulfone derivatives by treatment of DMSO or hydrogen peroxide. Esters of phenylalanine,
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threonine, and tyrosine were also subjected to the protease-catalyzed oligomerization (62). Polymerization of L-glutamic acid diethyl ester hydrochloride took place in the presence of papain or α-chymotrypsin as catalyst to give the corresponding oligomer composed of 5–9 glutamic acid residues (63,64). An nmr analysis showed that the product consisted exclusively of α-peptide linkage (65). Diethyl L-aspartate was polymerized by alkanophilic protease from Streptomyces sp. to give poly(ethyl α,β-L-aspartate) with weight-average molecular weight (M w ) up to 3600 (66). The ratio of α-linkage was about 88%, independent of the enzyme concentration. In order to enhance the molecular weight, protease was modified by mutation technique to show high catalytic activity in an aqueous N,N-dimethylformamide (DMF) solution. A subtilisin mutant (subtilisin 8350) derived from BPN (subtilisin from Bacillus amyloliquefaciens) via six site-specific mutation (Met 50 Phe, Gly 169 Ala, Asn 76 Asp, Gln 206 Cys, Tyr 217 Lys, and Asp 218 Ser) induced the polymerization of L-methionine methyl ester in the aqueous DMF to produce poly(methionine) with DP up to 50 (67). The increase of the molecular weight is due to the improvement of the product solubility and minimization of the enzymatic peptide cleavage under the high concentration of DMF. Another mutant (subtilisin 8397) showing higher stability in DMF, the same as 8350 except that there is no change for Tyr 217, has been applied as the catalyst for the polymerization of single amino acid, dipeptide, and tripeptide methyl esters (68). A different type of peptide hydrolase, dipeptidyl transferase (dipeptidylpeptide hydrolase), catalyzed the polymerization of dipeptide amide in an aqueous solution. In the case of glycyl-L-phenylalaninamide, trimer was formed in 78% yield (69). The polymerization of glycyl-L-tyrosinamide produced the corresponding oligomer with DP up to 8 (70). Protease was used as catalyst for polymer modification. Phenylalanine residues at the side chain of methacrylamide polymers were coupled with alanine t-butyl ester by α-chymotrypsin catalyst in water–chloroform solvent (71). Up to 35% peptide-bond formation was achieved for 7 days at room temperature. Polyamide synthesis was performed by cellulase-assisted polycondensation of chiral fluorinated compound having carboxylic acid and amino groups (72).
Polyesters Syntheses of aliphatic polyesters by fermentation and chemical processes have been extensively studied in a viewpoint of biodegradable materials. Recently, another approach of their production has been performed by using an isolated lipase or esterase as catalyst via nonbiosynthetic pathways under mild reaction conditions. Lipase and esterase are enzymes which catalyze hydrolysis of esters in an aqueous environment in living systems. Some of them can act as a catalyst for the reverse reactions, esterifications and transesterifications, in organic media (1–5). These catalytic actions have been expanded to enzymatic synthesis of polyesters. Figure 5 represents three major reaction types of lipase-catalyzed polymerization leading to polyesters (6–14,73).
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Fig. 5. Typical routes of polyester production using an isolated enzyme as catalyst.
Polymerization of Oxyacids and Their Esters. Oxyacids. In 1985, a lipase-catalyzed polymerization
of 10hydroxydecanoic acid was reported. The monomer was polymerized in benzene using poly(ethylene glycol) (PEG)-modified lipase soluble in the medium to give an oligoester with DP more than 5 (74). Ricinoleic acid, 12-hydroxyoctadecanoic acid, 16-hydroxyhexadecanoic acid, and 12-hydroxydecanoic acid were polymerized by lipase from Candida cylindracea (lipase CC) or Chromobacterium viscosum as a catalyst at 35◦ C in water, hydrocarbons, or benzene (75). The molecular weight of the polymers was ca 1×103 . Oligomerization of ricinoleic acid proceeded in the presence of lipase CC immobilized on ceramics (76). In the polycondensation of 10-hydroxydecanoic and 11-hydroxyundecanoic acids, a large amount of lipase CC catalyst (10 weight fold for the monomer) afforded the corresponding polyesters with relatively high molecular weight (77, 78). From the latter monomer, the polymer with M w of 2.2×104 was formed in the presence of activated molecular sieves. Porcine pancreas lipase (PPL) polymerized 3-hydroxybutyric and 12hydroxydodecanoic acids in anhydrous hydrophobic solvents (79). The molecular weight of the polymer from the former was low (ca 500), whereas the polymerization of the latter at 75◦ C produced the polymer with molecular weight of 3×103 . A cellulase-assisted polymerization of chiral fluorinated compound having carboxylic acid and phenolic groups produced the aromatic polyester (72). Enzymatic synthesis of a methacrylamide-type polyester macromonomer was reported (80,81). In the polymerization of 12-hydroxydodecanoic acid in the presence of 11-methacryloylaminoundecanoic acid using lipase CC or Candida antarctica lipase (lipase CA) as catalyst, the polymerizable group was quantitatively incorporated into terminal of the polymer chain. By using characteristic catalysis of lipase, regio- and enantioselective polymerizations of oxyacids have been achieved. Lipase CA catalyzed regioselective
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polymerization of cholic acid, in which the hydroxy group at 3-position was regioselectively acylated to give the oligoester with molecular weight less than 1×103 (82). An optically active oligoester was obtained by the enantioselective polymerization of racemic 10-hydroxyundecanoic acid catalyzed by lipase CC. The resulting polymer was enriched in the (S) enantiomer with 60% enantiomeric excess (ee) and the (R)-enriched unreacted monomer with 33% ee was recovered (83). In the polymerization of racemic lactic acid catalyzed by lipase CA at 50◦ C (84), nonamer was detected in the product by MALDI-TOF mass measurement. A hplc analysis showed that the D-enantiomer possessed higher enzymatic reactivity. Oxyacid Esters. The polymerization of ethyl glycolate using PEG-modified esterase from hog liver and lipase from Aspergillus niger (lipase A) gave oligo(glycolic acid) with DP up to 5 (85). PPL catalyzed the polymerization of methyl esters of 5-hydroxypentanoic and 6-hydroxyhexanoic acids (86). In the polymerization of the latter in hexane at 69◦ C for more than 50 days, the polymer with DP up to 100 was formed. Relationships between solvent type and polymerization behaviors were systematically investigated; hydrophobic solvents such as hydrocarbons and diisopropyl ether were suitable for the enzymatic production of high molecular weight polymer. Polycondensation of various hydroxyesters, ethyl esters of 3- and 4-hydroxybutyric acids, 5- and 6-hydroxyhexanoic acids, 5-hydroxydodecanoic acid, and 15-hydroxypentadecanoic acid, proceeded by Pseudomonas sp. lipase catalyst to give the corresponding polyesters with molecular weight of several thousands (87). A symmetrical hydroxy diester, dimethyl β-hydroxyglutarate, was enantioselectively polymerized by lipase catalyst to produce a chiral oligomer (dimer or trimer) with 30–37% ee (88). The enantioselective polymerization of ε-substitutedε-hydroxy esters took place in the presence of PPL catalyst, yielding optically active oligomers (DP < 6) (89). The enantioselectivity increased as a function of bulkiness of the monomer substituent. Optically active polyesters with molecular weight more than 1×103 were obtained by the copolymerization of the racemic oxyacid esters with methyl 6-hydroxyhexanoate.
Lipase-Catalyzed Polymerization of Dicarboxylic Acids or Their Derivatives. Enzymatic synthesis has been achieved via various combinations of dicarboxylic acid derivatives and glycols. As to the diacid monomer, dicarboxylic acids, activated and nonactivated esters, cyclic acid anhydrides, and polyanhydrides were enzymatically reacted with glycols under mild reaction conditions. Dicarboxylic Acids. Immobilized Mucor miehei lipase (lipase MM) catalyzed polycondensation of adipic acid and 1,4-butanediol by using a horizontal two-chamber reactor in the presence of molecular sieves as dehydrating agent (90). A low dispersity polyester with DP=20 was obtained by the two-stage polymerization. The polymerization of dicarboxylic acids and glycols proceeded by using lipase CA catalyst in a solvent-free system, despite the initial heterogeneous mixture of the substrates (91–94). The polymerization behaviors strongly depended on the chain length of both monomers (93). The polymerization under reduced pressure increased the molecular weight of polyesters. The detailed studies in the combination of adipic acid (A) and 1,4-butanediol (B) showed that the propagation took place by the reaction of the preliminary adduct (AB) with a hydroxyterminated species (92).
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For the solvent-free polycondensation, a small amount of adjuvant was effective for the polymer production when both monomers were solid at the reaction temperature (93). In the polymerization of adipic acid and 1,6-hexanediol, loss of the enzymatic activity was small during the polymerization, whereas less than half of the activity remained in using glycols with methylene chain length less than 4 (94). An attempted experiment allowed the polyester production from adipic acid and 1,6-hexanediol in 200-kg scale. This solvent-free system has a good potential as an environmentally friendly practical synthetic process of polymeric materials owing to the mild reaction conditions without using organic solvents and toxic catalysts. A dehydration polymerization of dicarboxylic acids and glycols took place by lipase catalyst even in water (95,96). This catalysis of lipase is quite specific since a dehydration reaction in an aqueous solution is generally disfavored by water, which is in equilibrium with starting materials because of the law of mass action. Hydrophobic monomer combinations gave the polyesters in good yields. Lipase CA catalyzed the polymerization of adipic acid and glycerol to give the oligomeric products (97). The presence of molecular sieves improved the molecular weight. The molecular weight increase was achieved using vacuum system, which removed the resulting water molecules during the polymerization (98,99). An aliphatic polyester with M w of 4.2×104 was obtained from sebacic acid and 1,4butandiol using lipase MM catalyst in diphenyl ether at 37◦ C for 7 days under the reduced pressure. The molecular weight was much larger than that obtained under ambient pressure. The polymerization of isophthalic acid and 1,6-hexanediol at 70◦ C produced the corresponding aromatic polyesters with M w of 5.5×104 (100). Dicarboxylic Acid Diesters. Since unactivated esters, typically alkyl esters, show low reactivity toward lipase catalyst, the polycondensation with glycols was often performed under vacuum to produce polyesters of high molecular weight. Lipase MM-catalyzed polycondensation of diethyl sebacate and 1,4-butanediol under vacuum produced the polymer with M w more than 2×104 (98). There is, of course, an equilibrium between the monomers and polymer in the lipase-catalyzed polycondensation of dialkyl esters and glycols. In the lipase CC- or MM-catalyzed polymerization of dimethyl succinate and 1,6-hexanediol in toluene, adsorption of methanol by molecular sieves or elimination of methanol by nitrogen bubbling shifted the thermodynamic equilibrium (101). When dicarboxylic acid dialkyl esters and α,ω-alkylene glycols were used as monomers, cyclic oligomers were formed from any monomer combinations examined (102). The yield of the cyclics depended on the monomer structure, initial concentration of the monomers, and reaction temperature. The ring-chain equilibrium was observed and the molar distribution of the cyclic species obeyed the Jacobson–Stockmayer equation. Activated esters of halogenated alcohols, such as 2-chloroethanol, 2,2,2trifluoroethanol, and 2,2,2-trichloroethanol, have often been used as substrate for enzymatic synthesis of esters (4), owing to the increase of the electrophilicity (reactivity) of the acyl carbonyl and the avoidance of significant alcoholysis of the products by decreasing the nucleophilicity of the leaving alcohols. Polymerization of bis(2,2,2-trichloroethyl) alkanediaoates with glycols proceeded by PPL catalyst in anhydrous solvents of low polarity to produce the
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Fig. 6. Lipase-catalyzed polycondensation of divinyl esters and glycols.
polyesters with molecular weight of several thousands (103,104). The oligomer formation was observed in the polymerization of bis(2-chloroethyl) succinate and 1,4-butanediol using Pseudomonas fluorescens lipase (lipase PF) as catalyst (105). Vacuum method was applied to shift the equilibrium forward by removal of the activated alcohol formed (98,99,106,107). In the polycondensation of bis(2,2,2trifluoroethyl) sebacate and aliphatic diols, lipases CC, MM, PPL, and Pseudomonas cepacia lipase (lipase PC) produced the polymer with M w more than 1×104 . Among the enzymes examined, lipase MM showed the highest catalytic activity (106). As to solvents, diphenyl ether and veratrole were suitable for the production of the high molecular weight polyesters under vacuum. In the PPLcatalyzed reaction of bis(2,2,2-trifluoroethyl) glutarate with 1,4-butanediol, the increase of the molecular weight was attained by periodical vacuum using veratrole or 1,3-dimethoxybenzene as less-volatile solvent (107). In lipase-catalyzed transesterifications, enol esters have been used as acyl agents (4), since the leaving unsaturated alcohol irreversibly tautomerizes to an aldehyde or a ketone, leading to the desired product in high yields. Bis(enol ester)s were reported to be much effective for the enzymatic synthesis of polyesters under mild reaction conditions (Fig. 6) (108); the polymerization of divinyl adipate and 1,4-butanediol proceeded by lipase PF at 45◦ C, and adipic acid and diethyl adipate did not afford the polymeric materials under the similar reaction conditions. Various lipases (lipases CA, MM, PC, and PF) catalyzed the polycondensation of divinyl adipate or divinyl sebacate with α,ω-glycols with different chain length (109,110). A combination of divinyl adipate, 1,4-butanediol, and lipase PC afforded the polymer with number-average molecular weight (M n ) of more than 2×104 . The polymerization behaviors of the lipase-catalyzed polymerization of divinyl adipate and 1,4-butanediol have been widely investigated (111–114). During the polymerization, the hydrolysis of the terminal vinyl ester took place, resulting in the significant limitation of the formation of the polyester with high molecular weight. A mathematical model describing the kinetics of this polymerization was proposed, which effectively predicts the composition (terminal structure) of the polyester. Another irreversible approach was performed by using bis(2,3-butanedione monoxime) alkanedioates as diester substrate (115). The polymerization with α,ωalkylene glycols by lipase MM produced the polymer with M n up to 7.0×103 . An enantioselective polymerization of racemic substrates took place through lipase catalysis, yielding optically active oligoesters and polyesters. The polymerization of bis(2-chloroethyl) 2,5-dibromoadipate with excess of 1,6-hexanediol using lipase A catalyst produced optically active trimer and pentamer (116).
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Fig. 7. Enantioselective polymerization of epoxy-containing diester with 1,4-butanediol.
PPL-catalyzed polymerization of bis(2,2,2-trichloroethyl) trans-3,4epoxyadipate with 1,4-butanediol enantioselectively proceeded in anhydrous diethyl ether to give an optically active polyester with molecular weight of 5.3×103 (Fig. 7) (117). The molar ratio of the diester to the diol was adjusted to 2:1 so as to produce the (−) polymer with enantiomeric purity of >96%. Polymerization of divinyl esters with triols regioselectively took place by lipase CA catalyst to give the soluble polymers with M w of more than 1×104 (118,119). MALDI-TOF MS analysis confirmed the presence of a linear polyester with hydroxy substituents. An nmr analysis of the product obtained from divinyl sebacate and glycerol in bulk at 60◦ C showed that 1,3-diglyceride was a main unit and the branching unit (triglyceride) was contained in the resulting polymer. The regioselectivity of the acylation between primary and secondary hydroxy groups was 74:26. By choosing the reaction conditions, the polymer consisting exclusively of 1,3-acylated unit of glycerol was formed. Lipase CA catalyzed the regioselective polymerization of sugar alcohols such as sorbitol and mannitol with divinyl sebacate to give polyesters containing sugar group in the backbone (120). Some proteases show an esterase activity, especially in their catalytic activity for regioselective acylation of sugars. By utilizing this property, protease-catalyzed synthesis of sugar-containing polyesters was demonstrated (121). Polycondensation of sucrose with bis(2,2,2-trifluoroethyl) adipate using an alkaline protease from Bacillus sp. as catalyst proceeded to give the polymer (M n =1.6×103 ), which was claimed to have ester linkages at the C-6 and C-1 positions on the sucrose (Fig. 8). In using divinyl adipate as diester monomer, the molecular weight reached 1.1×104 (122). Another approach of enzymatic synthesis of sugar-containing polyesters was demonstrated (123). Lipase CA-catalyzed reaction of sucrose or trehalose with an excess of divinyl adipate produced 6,6 -diacylated product having vinyl esters at both ends, which was employed as monomer in the enzymatic polycondensation with various glycols, yielding linear polyesters with M w up to 2.2×104 . Unsaturated ester oligomers were synthesized by lipase-catalyzed polymerization of diesters of fumaric acid and 1,4-butanediol (124). Mild reaction conditions did not induce isomerization of the double bond to give all-trans oligomers showing crystallinity, whereas the industrial unsaturated polyester having a mixture of cis and trans double bonds is amorphous (125). The enzymatic polymerization of bis(2-chloroethyl) fumarate with xylylene glycol produced the unsaturated oligoester containing aromaticity in the backbone (126).
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Fig. 8. Enzymatic synthesis of sucrose-containing polyester.
An unsaturated polyester possessing exclusively cis structure was synthesized by lipase CA-catalyzed polymerization of dimethyl maleate and 1,6hexanediol in toluene (127). During the polymerization, formation of cyclic oligomers was observed. The cycles were semicrystalline, whereas the linear polymer was amorphous. In the lipase CA-catalyzed copolymerization of dimethyl maleate and dimethyl fumarate with 1,6-hexanediol, the content of the cyclization was found to mainly depend on the configuration and concentration of the monomers (128). Polyesters containing an aromatic moiety in the backbone were synthesized by lipase CA-catalyzed polymerization of dicarboxylic acid divinyl esters and glycols under mild reaction conditions. Divinyl esters of isophthalic acid, terephthalic acid, and p-phenylene diacetic acid were enzymatically polymerized with α,ωalkylene glycols to give the polymers with molecular weight of several thousands (129). Aromatic polyesters were also synthesized from methyl esters of terephthalic and isophthalic acids with 1,6-hexanediol in the presence of lipase CA (130). In using methyl isophthalate as monomer, macrocyclic compounds were formed as by-product. Protease from Bacillus licheniformis catalyzed the oligomerization of esters of terephthalic acid with 1,4-butanediol (131). Lipase-catalyzed synthesis of aromatic polyesters was achieved by the polymerization of divinyl esters with xylylene glycols (129,132). Enzymatic synthesis of fluorinated polyesters was demonstrated (133). Fluorinated diols such as 2,2,3,3-tetrafluoro-1,4-butanediol and 2,2,3,3,4,4-hexafluoro1,5-pentanediol were used as glycol substrate and polymerized with divinyl adipate using lipase CA catalyst. The enzymatic synthesis of polyester was also achieved in supercritical fluoroform solvent by the polymerization of bis(2,2,2trichloroethyl) adipate and 1,4-butanediol (134). The molecular weight increased as a function of the pressure. Anhydrides. Ring-opening poly(addition-condensation) of cyclic acid anhydrides with glycols proceeded through lipase catalysis (135). The polymerization of succinic anhydride with 1,8-octanediol proceeded using lipase PF catalyst at room temperature to produce the polyester with M n of 3×103 .
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Fig. 9. Cyclic monomers polymerized by lipases.
Polyanhydrides were effective as diacid substrate for enzymatic synthesis of polyesters (136). The reaction of poly(azelaic anhydride) and 1,8-octanediol took place by lipase CA catalyst to give the corresponding polyester with molecular weight of several thousands. In the reaction of poly(azelaic anhydride) and glycerol, a highly branched polyester was obtained. Oxiranes such as benzyl glycidate and glycidiyl phenyl ether were polymerized with succinic anhydride in the presence of PPL at 60 or 80◦ C (137,138). The reaction of succinic anhydride with serine residue of the lipase catalyst produces a carboxylic acid moiety, which might act as acid catalyst for ring-opening of oxirane. Ring-Opening Polymerization of Cyclic Esters. Polyester syntheses have been achieved by enzymatic ring-opening polymerization of cyclic esters with various ring-sizes. Figure 9 summarizes cyclic monomers so far polymerized through lipase catalysis. Lactones. Small-size (four-membered) lactone derivatives have been reported to be polymerized through lipase catalysis. The polymerization of βpropiolactone (β-PL) proceeded by using Pseudomonas family lipases as catalyst in bulk to give a mixture of linear and cyclic oligomers (139). By employing a very small amount of lipase CC (0.5 versus for the monomer), high molecular weight poly(β-PL) was formed (140). Ring-opening polymerization of racemic α-methyl-β-propiolactone using lipase PC catalyst proceeded enantioselectively to produce an optically active (S)enriched polymer (141). The highest ee value of the polymer was 0.50. An nmr analysis of the product showed that the stereoselectivity during the propagation resulted from the catalyst enantiomorphic-site control. β-Butyrolactone (β-BL) was enzymatically polymerized to give poly(βhydroxybutyrate) (PHB), which is a polyester produced in vivo by bacteria for an energy-storage substance. PPL-catalyzed polymerization of β-BL in bulk at
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room temperature produced PHB with molecular weight around 1×103 (142). In the polymerization at high temperature (80 or 100◦ C), PHB with higher molecular weight was obtained by lipase CC, PF, or PPL catalyst (143,144). A significant amount of the cyclic PHB fraction was formed and the content of the cycles increased with increasing the monomer conversion. Enantioselective polymerization of β-BL was achieved by using thermophilic lipase to give (R)-enriched PHB with 20–37% ee (145). PHB depolymerase is an enzyme catalyzing hydrolysis of PHB and its catalytic site is a serine residue, the same as lipase. The polymerization of β-BL proceeded using two types of PHB depolymerase with or without substrate-binding domains (SBD) as catalyst (146). The SBD-lacking PHB depolymerase showed higher catalytic activity. Chemoenzymatic synthesis of biodegradable poly(malic acid) was demonstrated by lipase-catalyzed polymerization of benzyl β-malolactone, followed by the debenzylation (147). The molecular weight of poly(benzyl β-malolactone) increased by the copolymerization with a small amount of β-PL (17 mol% for the monomer) (148). Five-membered unsubstituted lactone, γ -butyrolactone, is not polymerized by conventional chemical catalysts. However, oligomer formation from γ butyrolactone was observed by using PPL or Pseudomonas sp. lipase as catalyst (87,142). Medium size lactones, δ-valerolactone (δ-VL, six-membered) and εcaprolactone (ε-CL, seven-membered), were subjected to lipase-catalyzed polymerizations. Lipases CC, PF, and PPL showed high catalytic activity for the polymerization of δ-VL (149,150). The molecular weight of enzymatically obtained poly(δ-VL) was relatively low (less than 2×103 ). ε-CL was enzymatically polymerized by various lipases of different origin, lipases CA, CC, PC, PF, and PPL (86,149–157). Among them, lipase CA was the most active toward the ε-CL polymerization; a very small amount of lipase CA (less than 1 wt% for ε-CL) was enough to induce the polymerization (151). Under appropriate reaction conditions, the molecular weight reached more than 4×104 (157). In the lipase CA-catalyzed polymerization in organic solvents, cyclic oligomers were mainly formed, whereas the main product in the bulk polymerization was of linear structure (155). The detailed kinetics of the ε-CL polymerization showed that termination and chain transfer did not occur and the monomer consumption followed a firstorder rate law under appropriate conditions, indicating that the system provided controlled polymerizations where the molecular weight was a function of the monomer to initiator stoichiometry (152,153,156). Effect of reaction medium has been systematically investigated in the lipase CA-catalyzed polymerization of ε-CL (157). Solvents having log P values from −1.1 to 0.49 showed low propagation rates; on the other hand, solvents with log P values from 1.9 to 4.5 efficiently induced the polymerization, leading to high molecular weight polymer. The monomer-to-solvent ratio also affected the polymerization behaviors. Enzymatic hydrolytic degradation of poly(ε-CL) in toluene also took place using lipase CA catalyst to give oligomers with molecular weight of less than 500 (158). After the removal of the solvent from the reaction mixture, the residual
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oligomer was polymerized in the presence of the same catalyst of lipase. From these data is proposed a basic concept that the degradation–polymerization could be controlled by presence or absence of the solvent, providing a new methodology of plastics recycling. Substituted medium size lactones were polymerized by lipase catalyst. Ring-opening polymerization of α-methyl-substituted six- and seven-membered lactones (α-methyl-δ-valerolactone and α-methyl-ε-caprolactone, respectively) proceeded using lipase CA catalyst in bulk (159). As to (R)- and (S)-3-methyl-4-oxa6-hexanolides (MOHELs), lipase PC induced the polymerization of both isomers. The apparent initial rate of the S isomer was seven times larger than that of the R isomer, suggesting that the enantioselective polymerization of MOHEL took place through lipase catalysis (160). Poly(1,4-dioxane-2-one) is a biocompatible polymer with good flexibility and tensile strength for medical applications. Metal-free poly(1,4-dioxane-2-one) with M w up to 4.1×104 was synthesized by lipase CA-catalyzed ring-opening polymerization of 1,4-dioxan-2-one (161). Lipase-catalyzed ring-opening polymerization of nine-membered lactone, 8octanolide (OL), has been reported (162). Lipases CA and PC showed the high catalytic activity for the polymerization. Four unsubstituted macrolides, 11-undecanolide (12-membered, UDL) (163, 164), 12-dodecanolide (13-membered, DDL) (164,165), 15-pentadecanolide (16membered, PDL) (163,164,166,167), and 16-hexadecanolide (17-membered) (168), were subjected to the lipase-catalyzed polymerization. An nmr analysis showed that the terminal structure of the polymer obtained in bulk was of carboxylic acid at one end and of alcohol at the other terminal. The bulk polymerization of PDL using lipase CA or MM as catalyst produced the corresponding polyester with high molecular weight up to 3.4×104 (167). The polymerization behaviors (rate of the monomer consumption and molecular weight of the polymer) depended on the water content in the reaction system. Enzymatic ring-opening polymerization of macrolides (UDL, DDL, and PDL) proceeded even in an aqueous medium (169). The enzymatic polymerization of lactones is explained by considering the following reactions as the principal reaction course (Fig. 10) (160,163,170,171). The key step is the reaction of the lactone with lipase involving the ring-opening of the lactone to give the acyl-enzyme intermediate (enzyme-activated monomer, EM). The initiation is a nucleophilic attack of water, which is probably contained in the enzyme, onto the acyl carbon of the intermediate to produce ω-hydroxycarboxylic acid (n = 1, the shortest propagating species). In the propagation stage, the intermediate is nucleophilically attacked by the terminal hydroxyl group of a propagating polymer to produce a one-unit-more elongated polymer chain. This is a monomer-activated mechanism in contrast to an active chain-end mechanism, the widely known polymerization mechanism. Macrolides have virtually no ring strain, and hence, show similar reactivities with acyclic fatty acid alkyl esters in the alkaline hydrolysis and lower anionic ring-opening polymerizability than ε-CL. However, polymerization of the macrolides using lipase PF catalyst proceeded much faster than that of ε-CL. This specific polymerizability by lipase catalyst was quantitatively evaluated by Michaelis–Menten kinetics (160,168,170–172). For unsubstituted lactones in the
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Fig. 10. Postulated mechanism of lactone polymerization catalyzed by lipase.
range of ring-size from 7 to 17, linearity was observed in the Hanes–Woolf plot for the formation of the acyl-lipase intermediate, indicating that the polymerization followed Michaelis–Menten kinetics. V max(lactone) /K m(lactone) and V max(lactone) values increased as a function of the ring-size; on the other hand, K m(lactone) values were not so different from each other. These data imply that the enzymatic polymerizability increased as the ring-size increased, and the large polymerizability of macrolides through lipase catalysis is mainly due to the large reaction rate (V max ), but not to the binding abilities, ie, the ring-opening reaction process of the lipase–lactone complex to the acyl-enzyme intermediate is the key step of the polymerization. Fluorinated lactones in the ring-size from 10 to 14 were enantioselectively polymerized using lipase catalyst (173). The lipase CA-catalyzed polymerization of 10-fluorodecan-9-olide (10-membered) produced the optically active polymer with positive rotation. Interestingly, the corresponding oxyacid gave an optically inactive polyester. Enzymatic synthesis of aliphatic ester copolymers was achieved by lipasecatalyzed polymerization of two lactones. The copolymerization of δ-VL and εCL catalyzed by lipase PF produced the corresponding copolymer having random structure of both units (174). In the copolymerization of OL with ε-CL or DDL, random copolyesters were also formed (162), suggesting the frequent occurrence of transesterifications between the polyesters. On the other hand, the copolymer from ε-CL and PDL was not statistically random (166). Polyesters with high optical purity were synthesized by the lipase CAcatalyzed copolymerization of racemic β-BL with ε-CL or DDL (175). (S)-β-BL was preferentially reacted with DDL to give the (S)-enriched optically active copolymer with ee of β-BL unit = 69%. δ-CL was also enantioselectively copolymerized by the lipase catalyst to give the (R)-enriched optically active polyester with ee up to 76%. Frequent occurrence of transesterification between polyesters chains was expanded to synthesis of random ester copolymers by the lipase-catalyzed
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polymerization of lactones in the presence of poly(ε-CL) (176). Intermolecular transesterifications between poly(ε-CL) and poly(PDL) also took place through lipase catalysis. Ester copolymers were synthesized by lipase-catalyzed copolymerization of lactones, divinyl esters, and glycols (177). The 13 C nmr analysis showed that the resulting product was not a mixture of homopolymers, but a copolymer derived from the monomers, indicating that two different modes of polymerization, ringopening polymerization and polycondensation, simultaneously take place through enzyme catalysis in one pot to produce ester copolymers. Immobilized lipase showing high catalytic activity toward the enzymatic synthesis of polyesters was demonstrated (178). Only a small amount of immobilized lipase PF adsorbed on a Celite was effective for the polymerization of lactones. The catalytic activity was further enhanced by the presence of a sugar or PEG at the immobilization. Surfactant-coated lipase efficiently catalyzed the ring-opening polymerization of lactones in organic solvents, in which the modified enzyme was soluble (179). Enzymatic synthesis of end-functionalized polymers such as macromonomers and telechelics has been achieved by initiator and terminator methods. An alcohol could initiate the ring-opening polymerization of lactones by lipase catalyst (“initiator method”). In the lipase CA-catalyzed polymerization of DDL using 2-hydroxyethyl methacrylate as initiator, the methacryloyl group was quantitatively introduced at the polymer terminal, yielding the methacryl-type polyester macromonomer (180). In the lipase-catalyzed polymerization of ε-CL in the presence of functional alcohols (181), end-functionalized poly(ε-CL) as well as the cyclic by-product was formed. Polyesters bearing the sugar moiety at the polymer terminal was synthesized by lipase CA-catalyzed polymerization of ε-CL in the presence of alkyl glucopyranosides (182–184). In the initiation step, the primary hydroxy group of the glucopyranoside was regioselectively acylated. Poly(ε-CL) monosubstituted first generation dendrimer was synthesized using lipase CA as catalyst. The monoacylation of the initiator took place at the initial stage (185). Polymeric hydroxy group also initiated the enzymatic ring-opening polymerization of ε-CL (186). The polymerization was performed using thermophilic lipase as catalyst in the presence of hydroxyethyl cellulose (HEC) film to produce HECgraft-poly(ε-CL) with degree of substitution from 0.10 to 0.32. Single-step synthesis of polyester macromonomers was achieved by lipasecatalyzed polymerization of lactones in the presence of vinyl esters acting as terminator (“terminator method”) (187,188). A methacryl-type poly(DDL) macromonomer was obtained using vinyl methacrylate (12.5 or 15 mol% based on DDL) and lipase PF as terminator and catalyst, respectively. By the addition of divinyl sebacate, the telechelic polyester having a carboxylic acid group at both ends was synthesized. Cyclic Diesters. Cyclic diesters were subjected to the lipase-catalyzed ringopening polymerization. Lactide, cyclic dimer of lactic acid, was polymerized by lipase PC in bulk at high temperature (80–130◦ C) to produce poly(lactic acid) with M w up to 2.7 × 105 (189,190). Protease (proteinase K) also induced the polymerization of lactide; however, the catalytic activity was relatively low.
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Ring-opening polymerization of cyclic diesters obtained from diacid and glycol, ethylene dodecanoate, ethylene tridecanoate, and 1,4,7-trioxa-cyclotridecane8,13-dione, took place through lipase catalysis (191,192). The former two monomers were polymerized by lipase CA, PC, or PF catalyst (191). The enzyme origin affected the polymerization behaviors; the polymerization of these bislactones using lipase PC catalyst proceeded faster than that of ε-CL and DDL, whereas the reactivity of these cyclic diesters was in the middle of ε-CL and DDL in using lipase CA.
In Vitro PHA Polymerase-Catalyzed Polymerization to PHA. Alcaligenes eutrophus has been used for industrial production of poly(hydroxyalkanoate)s (PHAs). PHA is prepared from acetyl CoA in three steps and the last step is the chain growth polymerization of hydroxyalkanoate CoA esters catalyzed by PHA polymerase, yielding PHA of high molecular weight, which has been in vitro examined, leading to synthesis of PHAs with well-defined structure. This synthetic process obeys the biosynthetic pathways (see POLY(3-HYDROXYALKANOATES)). The growing polymer chain was covalently attached to a highly conserved cysteine residue (Cy319) of the polymerase (193). The granules of the precipitated polymer were quickly formed when the purified polymerase was exposed to (R)-hydroxybutyryl CoA (HBCoA) (194,195). The artificial PHB granules were spherical with diameters of up to 3 µm, significantly larger than the native ones. The polymerization of the CoA monomers of (R)-hydroxyalkanoate was of chain growth in a living fashion; each molecule of the polymerase initiated and catalyzed the formation of one molecule of the polymer (196–198). By utilizing this property of polymerase, random and block copolyesters were synthesized. The resulting polymer had high molecular weight (>106 ). In the polymerization of racemic HBCoA, only the R monomer was polymerized. Furthermore, the presence of the S monomer did not reduce the polymerization rate of the R isomer. These data indicate that the S monomer does not act as competitive inhibitor for the polymerase. Recombinant PHA synthase from Chromatium vinosum showed different catalytic behaviors in comparison with that of A. eutrophus (199). In combination of this synthase with purified propionyl-CoA transferase of Clostridium propionicum, a two-enzyme in vitro PHB biosynthesis system was established, which allowed the PHB synthesis from (R)-hydroxybutyric acid as substrate (200). Hydrolase-Catalyzed Modification of Polymers. Terminal hydroxy group of poly(ε-CL) was reacted with carboxylic acids using lipase CA catalyst to give end-functionalized polyesters (181). Lipase MM catalyzed the regioselective transesterification of the terminal ester group of oligo(methyl methacrylate) with allyl alcohol (201). The lipase-catalyzed acetylation of high molecular weight methacrylic polymers containing racemic hydroxy groups in the side chain was achieved with a maximum conversion of 40% (202,203). The optical rotation was low (up to −1.2◦ ), suggesting a low enantioselectivity of this esterification. The enzymatic transesterification of amylose film with vinyl caprate in the isooctane solution containing solubilized subtilisin Carlsberg produced an amylose derivative regioselectively acylated at the C-6 position (204).
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Enzymatic epoxidation of polybutadiene was demonstrated (205). Lipase CA catalyzed the oxidation of polybutadiene using hydrogen peroxide as oxidizing agent in the presence of acetic acid.
Polycarbonates Polycondensation. Oligocarbonate with molecular weight of less than 1×103 was formed by lipase CC-catalyzed polycondensation of carbonic acid diphenyl ester with bisphenol A (206). Diethyl carbonate was polymerized with 1,4-butanediol by lipase CA catalyst (207,208). The successive two-step polymerization, the prepolymerization under ambient pressure, followed by the polymerization under vacuum (0.5 mm Hg), produced poly(tetramethylene carbonate) with M w of more than 4×104 . Activated dicarbonate, 1,3-propanediol divinyl dicarbonate, was used as new monomer for enzymatic synthesis of polycarbonates (209). Lipase CA catalyzed the polymerization with α,ω-alkylene glycols under mild reaction conditions and the M w value reached 8×103 . Aromatic polycarbonate was enzymatically obtained from the activated dicarbonate and xylylene glycol (132). Ring-Opening Polymerization. 1,3-Dioxan-2-one, six-membered cyclic carbonate, was polymerized in the presence of lipase catalysts (210–212). Under mild reaction conditions (≤70 ◦ C), lipase CA efficiently catalyzed the polymerization to give the corresponding polycarbonate with M n more than 1×104 (211,212). No ether bond was observed in the nmr spectrum of the product, indicating that elimination of carbon dioxide did not occur during the enzymatic polymerization. The polymerization in the presence of a small amount of PPL (0.1 or 0.25 wt% for the monomer) at 100◦ C produced the high molecular weight polymer (M w = 1.6×105 ) (210). The enzymatic polymerization of 5-methyl-5-benzyloxycarbonyl-1,3-dioxan2-one produced the corresponding polycarbonate (213). Lipases PF and CA showed high catalytic activity for the polymerization. Debenzylation by catalytic hydrogenation led to the water-soluble polycarbonate with pendent carboxyl group. Lipase CA catalyzed the polymerization of cyclic dicarbonates, cyclobis(hexamethylene carbonate) and cyclobis(diethylene glycol carbonate), to give the corresponding polycarbonates (214). The enzymatic copolymerization of cyclobis(diethylene glycol carbonate) with DDL produced a random ester-carbonate copolymer. Enzymatic synthesis of poly(ester-carbonate) was also achieved by the copolymerization of 1,3-dioxan-2-one and lactide (215). The PPL-catalyzed copolymerization at 100◦ C produced the copolymer with M w higher than 2×104 . Besides polyesters and polycarbonates, lipase-catalyzed synthesis of polymers from cyclic monomers has been reported. 3(S)-Isopropylmorpholine-2,5dione, six-membered depsipeptide, was polymerized by lipase PC and PPL catalysts to give poly(ester-amide) (216,217). High temperature (100 or 130◦ C) was required for the polymerization, yielding biodegradable poly(depsipeptide) with maximum M n = 3×104 . During the polymerization, the racemization of the valine residue took place. PPL-catalyzed synthesis of poly(phosphate) was demonstrated (218). The ring-opening polymerization of ethylene isopropyl phosphate,
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five-membered cyclic phosphate, took place at 40–100◦ C to give the polymer with molecular weight of ca 1×103 .
Polyaromatics In living cells, various oxidoreductases play an important role in maintaining the metabolism of living systems. So far, peroxidase containing Fe-active site, laccase containing Cu-active site, tyrosinase (polyphenol oxidase, Cu-active site), bilirubin oxidase (Cu-active site), etc, have been reported to act as catalyst for oxidative polymerization of phenol and aniline derivatives and for polymer modification via oxidative coupling.
Enzymatic Oxidative Polymerization. Polyphenols. For enzymatic oxidative polymerization of phenol derivatives, peroxidase has been often used as catalyst. Catalytic cycle of peroxidase is shown in Figure 11. Peroxidase catalyzes decomposition of hydrogen peroxide at
Fig. 11. Catalytic cycles of peroxidase for polymerization of phenols.
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Fig. 12. Peroxidase-catalyzed oxidative polymerization of phenol.
the expense of aromatic proton donors in living cells. In some cases, the peroxidasecatalyzed oxidation of these donors, eg, phenols, yields water-insoluble polymeric materials, which had not been characterized yet. In 1987, enzymatic synthesis of a new class of polyphenols has been first reported. An oxidative polymerization of p-phenylphenol using horseradish peroxidase (HRP) as catalyst was carried out in a mixture of water and water-miscible solvents such as 1,4-dioxane, acetone, DMF, and methyl formate to give powdery polymeric materials (219). The reaction medium composition greatly affected the molecular weight, and the highest molecular weight (2.6×104 ) was achieved in 85% 1,4-dioxane. In the case of phenol, the simplest and most important phenolic compound in industrial fields, conventional polymerization catalysts afford an insoluble product with noncontrolled structure since phenol is a multifunctional monomer for oxidative polymerization. On the other hand, the peroxidase catalysis induced the polymerization in an aqueous organic solvent to give a powdery polymer consisting of phenylene and oxyphenylene units showing relatively high thermal stability (Fig. 12) (220–224). HRP and soybean peroxidase (SBP) were active as catalyst for the polymerization in the aqueous 1,4-dioxane (220–222). However, the resulting polymer showed low solubility; the polymer was partly soluble in DMF and dimethyl sulfoxide, and insoluble in other common organic solvents. The solubility was much improved by using a mixed solvent of buffer and methanol, producing the DMF-soluble polymer with molecular weight of 2100–6000 in good yields. Furthermore, the unit ratio (regioselectivity) could be controlled by changing the solvent composition; the polymer in the range of the phenylene unit from 32 to 66% was obtained (223,224). So far, various phenol derivatives have been polymerized through peroxidase catalysis in the aqueous organic solvent (225–227). For the case of a combination of p-n-alkylphenols and HRP, the polymer yield increased as the chain length of the alkyl group increased from 1 to 5 (228,229). Polymer formation was observed in using all cresol isomers by HRP catalyst (230). The polymer was obtained in a high yield from p-i-propylphenol, whereas ortho and metaisomers were not polymerized under the similar reaction conditions. Poly(p-n-alkylphenol)s prepared in the aqueous 1,4-dioxane showed low solubility toward common organic solvents, and the molecular weight was in the range of several thousands. On the other hand, soluble oligomers with molecular weight less than 1000 were formed in using an aqueous DMF as solvent (231). Enzymatically synthesized polyphenols showed biodegradability (232), although the degradation rate was not high. Antioxidant effects of the polymers obtained from various phenols through the enzyme catalysis were evaluated (233). Pronounced improvement for the autooxidation of tetralin was observed.
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As to meta-alkyl substituted phenols, the soluble polyphenols were obtained by HRP or SBP catalyst in the aqueous methanol (234). Enzymatically synthesized poly(m-cresol) had glass-transition temperature (T g ) of higher than 200◦ C. The enzyme origin strongly influenced the polymer yield; HRP could readily polymerize the monomer having a small substituent, whereas in the case of large substituent monomers, the high yield was achieved by using SBP as catalyst. The enzymatic reaction kinetics on the HRP-catalyzed oxidation of p-cresol in aqueous 1,4-dioxane or methanol showed that the catalytic turnover number and Michaelis constant were larger than those in water (235). Numerical and Monte Carlo simulations of the peroxidase-catalyzed polymerization of phenols were demonstrated (236). The simulations predicted the monomer reactivity and polymer molecular weight, leading to synthesis of polymers with specific molecular weight and index. In an aqueous 1,4-dioxane, the formation of monomer aggregate was observed (237), which might elucidate the specific polymerization behaviors in such a medium. Effects of the monomer substituent and substituted position on the initial reaction rate in the HRP-catalyzed polymerization of substituted phenols were examined (238). Substrates with the electron-drawing group or ortho-substituted substrates showed low polymerizability. Lactoperoxidase also showed catalytic activity for the polymerization of phenols. Four interfacial systems, micelles, reverse micelles, a biphasic, and Langmuir trough systems, have been examined for preparation of the enzymatic synthesis of polyphenols. In the polymerization in micelle solution consisting of surfactant and buffer, the obtained polymer from p-phenylphenol had narrow molecular weight distribution in comparison of that in the aqueous 1,4-dioxane (239). HRP and p-ethylphenol were encapsulated in the reverse micelle, which was a ternary system composed of isooctane, water, and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). The introduction of hydrogen peroxide into the system induced the polymerization to produce the polymer particles in the diameter range from 0.1 to 2 µm quantitatively (240–242). Similar particles were obtained by pouring the solution of enzymatically prepared polyphenol into a nonsolvent containing AOT (243). HRP-catalyzed polymerization of p-alkylphenols proceeded in a biphasic system consisting of two mutually immiscible phases (isooctane and water) (226). The molecular weight increased as a function of the carbon number of the alkyl group. Enzymatic polymerization of phenol derivatives in a monolayer form was demonstrated (241,244,245). A monolayer was formed from p-tetradecyloxyphenol and phenol at the air–water interface in a Langmuir trough, which was polymerized by HRP catalyst in the subphase. The polymerized film could be deposited on silicon wafer with a transfer ratio of 100% for the Y-type film. The monolayer thickness determined by Eppipsometric and AFM was 27.8 ˚A. Poly(2,6-dimethyl-1,4-oxyphenylene) [poly(phenylene oxide), PPO] is widely used as high-performance engineering plastics, since the polymer has excellent chemical and physical properties, eg, a high T g (ca 210◦ C) and mechanically tough property. PPO was first prepared from 2,6-dimethylphenol monomer using a copper/amine catalyst system (246,247). 2,6-Dimethylphenol was also polymerized through HRP catalysis to give the polymer consisting of exclusively 1,4oxyphenylene unit (248). On the other hand, a small amount of Mannich-base
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Fig. 13. Enzymatic synthesis of PPO derivative from syringic acid.
and 3,5,3 5 -tetramethyl-4,4 -diphenoquinone units are contained in commercially available PPO. The polymerization also proceeded in the presence of laccase derived from Pycnoporus coccineus under air without the addition of hydrogen peroxide. HRP, SBP, and laccase catalysis induced a new type of oxidative polymerization of 4-hydroxybenzoic acid derivatives, 3,5-dimethoxy-4-hydroxybenzoic acid (syringic acid) and 3,5-dimethyl-4-hydroxybenzoic acid. The polymerization involved elimination of carbon dioxide and hydrogen from the monomer to give PPO derivatives with molecular weight up to 1.8×104 (Fig. 13) (110,249–251). Polymerization of p-alkoxyphenols regioselectively proceeded by HRP catalyst to give PPO (252). Peroxidase-catalyzed synthesis of poly(catechol) was achieved and the iodine-labeled polymer showed low electrical conductivity in the range of 10 − 6 –10 − 9 S·cm − 1 (253). Thiol-containing polyphenol was synthesized by peroxidase-catalyzed copolymerization of p-hydroxythiophenol and p-ethylphenol in reverse micelles (254). CdS nanoparticles were attached to the copolymer to give polymer–CdS nanocomposites. By a similar procedure, polyphenol–iron oxide composites were synthesized (242). The reverse micellar system was also effective for the enzymatic synthesis of poly(2-naphthol) showing a fluorescence characteristic of the naphthol chromophore (255). nmr, ir, and uv analyses showed the formation of the polymer with quinonoid structure. Bilirubin oxidase (BOD), a copper-containing oxidoreductase, catalyzed the oxidative polymerization of 1,5-dihydroxynaphthalene to give the polymer showing low solubility (256,257). The polymerization proceeded regioselectively to produce the polymer film having a long π -conjugated structure. This monomer was also polymerized by HRP catalyst (258). The polymerization in the presence of porous silicon (PS) wafer produced the polyphenol–PS composite showing optoelectronic properties. Bisphenol A was polymerized by SBP catalyst to give a soluble polymer with molecular weight of several thousands in good yields (259). Interestingly, the polymer was subjected to thermal curing at 150–200◦ C. 4,4 -Biphenol was polymerized by HRP catalyst in an aqueous 1,4-dioxane to give the polymer showing high thermal stability (260). The mechanistic study of the HRP-catalyzed oxidative polymerization was performed by using nmr spectroscopy (261,262). In the initial stage of the polymerization of 8-hydroxyquinoline-5-sulfonate, the oxidative coupling took place at carbons of the 2-, 4-, and 7-positions of the monomer. Polymerization and copolymerization of 8-hydroxyquinoline also took place through HRP catalysis (263).
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Fig. 14. Chemoenzymatic synthesis of poly(hydroquinone).
Peroxidase catalysis induced the oxidative polymerization of glucose-β-Dhydroquinone (arbutin) in a buffer to produce a water-soluble polyphenol (264). The acid treatment of the polymer led to the quantitative deglycosylation of the polymer, yielding poly(hydroquinone) soluble in polar organic solvents (Fig. 14). The resulting polymer was used as a mediator for amperometric glucose sensors (265). Another route for chemoenzymatic synthesis of poly(hydroquinone) from 4hydroxyphenyl benzoate was demonstrated (266), whose structure was different from that obtained from arbutin. Enzymatically synthesized phenolic copolymer containing fluorophore (fluorescein or calcein) was applied as array-based metalion sensor (267). A polynucleoside with unnatural polymeric backbone was synthesized by SBP-catalyzed oxidative polymerization of thymidine 5 -p-hydroxyphenylacetate (268). Chemoenzymatic synthesis of a new class of poly(amino acid), poly(tyrosine) containing no peptide bonds, was achieved by peroxidase-catalyzed oxidative polymerization of tyrosine ethyl esters, followed by alkaline hydrolysis (269). The amphiphile higher alkyl ester derivatives were also polymerized in micellar solution to give the polymer showing surface activity at the air–water interface (270). The polymerization was monitored by the quartz crystal microbalance (271). HRP catalysis induced a chemoselective polymerization of a phenol derivative having methacryloyl group (272). Only the phenol moiety was polymerized without involving vinyl polymerization of methacryloyl to give a polymer having the methacryloyl group in the side chain (Fig. 15). The resulting polymer was subjected to thermal and photochemical curings (273). A phenol with an acetylenic substituent in the meta position was also chemoselectively polymerized to give the polyphenol having the acetylenic group (274). The resulting polymer was converted to carbonized polymer in a much higher yield than enzymatically synthesized poly(m-cresol). Cardanol, a main component obtained by thermal treatment of cashew nut shell liquid, is a phenol derivative mainly having the meta substituent of a C-15 unsaturated hydrocarbon chain mainly with one to three double bonds. A new cross-linkable polymer was synthesized by the SBP-catalyzed polymerization of cardanol (110,251,275). The polymerization in an aqueous acetone produced oily polymeric materials having the carbon–carbon unsaturated group in the side chain. The curing by cobalt naphthenate gave the cross-linked film with high gloss surface. The hydrogenated cardanol derivative was also oxidatively polymerized by HRP (276).
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Fig. 15. Chemoselective polymerization of a phenol derivative having a methacryloyl group.
Fluorinated phenols, 3- and 4-fluorophenols and 2,6-difluorophenol, were subjected to peroxidase-catalyzed polymerization, yielding fluorine-containing polymerizations. During the polymerization, elimination of fluorine atom partly took place to give the polymer with complicated structure (277). Morphology of the enzymatically synthesized polyphenol was controlled under the selected reaction conditions. Monodisperse polyphenol particles in the submicron range were produced by HRP-catalyzed dispersion polymerization of phenol using poly(vinyl methyl ether) as stabilizer in an aqueous 1,4-dioxane (278– 280). The particle size could be controlled by the stabilizer concentration and solvent composition. Thermal treatment of these particles afforded uniform carbon particles. The particles were also formed from m-cresol and p-phenylphenol. Bienzymatic system (glucose oxidase + HRP) was used as catalyst for the polyphenol synthesis. This system induced the polymerization of phenol in the presence of glucose without the addition of hydrogen peroxide to produce the polymer in a moderate yield (281). Hydrogen peroxide was formed in situ by the oxidation of glucose catalyzed by glucose oxidase, which acted as oxidizing agent for the polymerization.
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In vitro synthesis of lignin, a typical phenolic biopolymer, was claimed by the HRP-catalyzed terpolymerization of lignin monomers, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (14:80:6 mol%) in an extremely dilute aqueous solution at pH 5.5 (282). Dialysis membrane method was applied to the polymerization of coniferyl and sinapyl alcohols, yielding insoluble polymeric materials (283). In the HRP-polymerization of coniferyl alcohol in the presence of a small amount of lignin component, the molecular weight distribution became much broader than that in the absence of lignin (284). Peroxidase-catalyzed polymerization behavior of coniferyl alcohol has been compared with that by laccase (285). Peroxidase oxidized the substrate faster than laccase in the presence of hydrogen peroxide. As to the laccase-catalyzed polymerization, the oxidation rate and reaction mechanism depended on the enzyme origin. Enzymatic polymerization of soluble lignin fragments (lignin oligomer) was demonstrated. In the polymerization catalyzed by HRP, or polyphenol oxidase (potato), brown precipitates were formed (286). The increase of the molecular weight was observed in the laccase-catalyzed treatment of the lignin oligomer (287). Peroxidase-catalyzed grafting of polyphenols on lignin has been attempted by HRP-catalyzed polymerization of p-cresol with lignin in the aqueous 1,4-dioxane or reverse micellar system. (288–290). The monomer was incorporated into lignin by the oxidative coupling between the monomer and the phenolic moiety of lignin. Low molecular weight coal (4 kDa) was polymerized by HRP or SBP catalyst in a mixture of DMF and buffer (291). The resulting product was partly soluble in DMF and the DMF-soluble part had a larger molecular weight than that of the starting substrate. A novel system of enzymatic polymerization, ie, a laccase-catalyzed crosslinking reaction of new “urushiol analogues” for the preparation of “artificial urushi,” has been demonstrated (Fig. 16) (292,293). Single-step synthesis of the urushiol analogues was achieved by using lipase as catalyst. These compounds were cured in the presence of laccase catalyst under mild reaction conditions without the use of organic solvents to produce the cross-linked polymeric film with high gloss surface and good elastic properties. Catechol derivatives directly
Fig. 16. Laccase-catalyzed curing of urushiol analogues to “artificial urushi.”
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connecting an unsaturated alkenyl group at 4-position of the catechol ring were also cured by laccase to give the cross-linked polymeric film showing ideal dynamic viscoelasticity (294). Polyanilines. Oxidoreductases also catalyze oxidative polymerization of aromatic amines. HRP induced the polymerization of aniline. In the HRPcatalyzed polymerization under neutral conditions, the polymer with complicated structure was obtained in low yields (295). The resulting polymer showed good third-order nonlinear optical properties (296). On the other hand, the polymerization using sulfonated polystyrene (SPS) as template produced the electroactive form of polyaniline (297–299). The resulting polymer was soluble in water and the conductivity reached 5×10 − 3 S·cm − 1 without doping. Besides SPS, a strong acid surfactant, sodium dodecylbenzenesulfonic acid, provided suitable local template environments leading to the formation of conducting polyaniline. Aniline was also polymerized by BOD catalyst to give the polyaniline film, which was electrochemically reversible in its redox properties in acidic solution (300). HRP-catalyzed oxidative polymerization of o-phenylenediamine in a mixture of 1,4-dioxane and phosphate buffer produced a soluble polymer consisting of an iminophenylene unit (301). From para and meta isomers, the polymer with well-defined structure was not obtained (302). Enzymatic polymer formation was observed from p-aminobenzoic acid (303), p-aminophenylmethylcarbitol (304), 2,5diaminobenzenesulfonate (305), and p-aminochalcones (306). Cytochrome c catalyzed oxidation of o-phenylenediamine to give oligomeric products (307). Monolayer of aniline/p-hexadecylaniline prepared by LB technique at the air–water interface was polymerized through HRP catalysis to give polymeric monolayer (244,245). A new class of polyaromatics was synthesized by peroxidase-catalyzed oxidative copolymerization of phenol derivatives with anilines. In case of a combination of phenol and o-pheneylenediamine, ftir analysis showed the formation of the corresponding copolymer (308). Polymer Modification by Oxidoreductases. Tyrosinase (polyphenol oxidase, a copper-containing monooxygenation enzyme) was used as catalyst for modification of chitosan. The enzymatic treatment of chitosan film in the presence of tyrosinase and phenol derivatives produced a new material of chitosan derivative (309). During the reaction, unstable o-quinones were formed, followed by the reaction with chitosan to give the modified chitosan. In the enzymatic treatment of p-cresol with a low concentration of chitosan (
