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Keywords:

  • enzymes;
  • esterification;
  • functionalization of polymers;
  • green polymer chemistry;
  • Michael addition

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information

The use of enzymes as catalysts for organic synthesis has become an increasingly attractive alternative to conventional chemical catalysis. Enzymes offer several advantages including high selectivity, ability to operate under mild conditions, catalyst recyclability, and biocompatibility. Although there are many examples in the literature involving enzymes for the synthesis of polymers, our search showed that very little had been done in the area of polymer modification. In this article, we will discuss enzyme catalysis in general and highlight our recent results concerning precision polymer functionalization using enzymatic catalysis—“green polymer chemistry.” © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2959–2976, 2009


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information

Enzymes are nature's catalysts that are designed to accelerate specific reactions taking place in the cell and its immediate surroundings. For example, lipases catalyze the hydrolysis of triglycerides into fatty acids and glycerols in vivo (Scheme 1).

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Scheme 1. Lipase-catalyzed hydrolysis of triglycerides.

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The function of enzymes, however, is not restricted to their natural, aqueous reaction medium; they are known to catalyze many synthetic transformations in organic solvents.1–4 In addition, they offer several advantages, such as high selectivity, ability to operate under mild conditions, catalyst recyclability, and biocompatibility, which render them environmentally friendly alternatives over conventional chemical catalysts.

Lipases are the most widely employed enzymes in organic reactions such as transesterification, esterification, aminolysis, and Michael addition.5–9Candida antarctica lipase B (CALB, Fig. 1) is a good catalyst for these transformations because of its high stability and reactivity.10

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Figure 1. 3D structure of Candida antarctica lipase B.8 (Gotor-Fernandez et al., Adv Synth Catal 2006, 348, 797–812, © Wiley-VCH Verlag GmbH and Co., reproduced by permission.)

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For example, comparison of the catalytic activities of different commercially available lipases in transesterification of vinyl acetate with n-octanol (Scheme 2) revealed that CALB was the most efficient lipase (Table 1).11

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Scheme 2. Lipase-catalyzed transesterification of vinyl acetate with n-octanol.

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Table 1. Comparison of the Specific Activity of Enzymes at 30 °C
EnzymeActivity (μmol min−1 mg−1)
Candida antarctica lipase B11.3
Lipozyme IM201.30
Pseudomonas lipase0.93
Candida cylindracea lipase0.00
Porcine pancreatic lipase0.00

Transesterification reactions are reversible, but when enolate esters are used, they liberate unstable enols as by-products, which instantly tautomerize to give the corresponding aldehydes (Scheme 3) or ketones. Vinyl esters are favored over isopropenyl esters because of less steric hindrance and thus higher reaction rates.6

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Scheme 3. Illustration of the mechanism of CALB-catalyzed transesterification of vinyl acetate with n-octanol.

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The amino acid sequence and active site structure of CALB have been resolved by Uppenberg et al.12 in 1994. It has a relatively narrow and deep active site consisting of serine (Ser105), histidine (His224), and aspartate (Asp187) as the catalytic triad. This active site is composed of two channels; one hosting the acyl- and the other hosting the alcohol-moiety of the substrate, with the first channel being the more spacious one.13 Therefore, the enzyme is expected to have a much higher degree of selectivity toward alcohol substrates. For example, although primary alcohols are generally excellent substrates for CALB, tertiary alcohols (e.g., t-butanol) are so inert toward CALB that they can be used effectively as solvents in CALB-catalyzed reactions.14 According to the well-elucidated catalytic mechanism,12, 15, 16 the transesterification of vinyl acetate with n-octanol (Scheme 2) consists of four subsequent steps (Scheme 3): First, a tetrahedral intermediate is formed by the nucleophilic attack of the hydroxyl group of serine to the carbonyl group of vinyl acetate, with the resulting oxyanion being stabilized by the so-called oxyanion hole via three hydrogen bonds, one with the glutamine (Gln106) and two with threonine (Thr40) units. In the second step, the ester bond is cleaved to release the alcohol product, that is, vinyl alcohol, and an acyl-enzyme complex is formed. In the third step, the reactant alcohol, that is, n-octanol, attacks the carbonyl group of the acyl-enzyme complex to form the second tetrahedral intermediate, which is again stabilized by the oxyanion hole. Finally, in the fourth step, the enzyme is deacylated to form the desired ester, octyl acetate. Scheme 3 helps to visualize the mechanism. The darker shaded part of the enzyme is meant to represent the acyl-hosting channel of the enzyme, while the lighter shaded half represents the alcohol “pocket.”

Enzymes can also catalyze Michael addition of nucleophiles to α,β-unsaturated carbonyl compounds. Kitazume et al.17 used lipases to catalyze Michael-type addition of amines and thiols to trifluorinated α,β-unsaturated carbonyl compounds. Lin and coworkers18 carried out a systematic study of Michael-type addition of imidazoles to acrylates in various organic solvents using several commercially available serine hydrolases. Gotor and coworkers19 demonstrated CALB-catalyzed Michael-type addition of secondary amines to acrylonitriles and proposed that the serine in the active site was not involved in the reaction. He suggested that the reaction mechanism involved the activation of the carbonyl group of the Michael-acceptor by the carbonyl hole, followed by histidine-aspartate-catalyzed proton transfer from the incoming nucleophile to the α-carbon of the Michael-acceptor (Scheme 4). Quantum-modeling of CALB-catalyzed Michael-type addition of thiols to α,β-unsaturated carbonyl compounds and the ability of a mutant of CALB, which lacked the serine in the active site, to catalyze the reaction20 were in agreement with the predictions made by Gotor et al.19

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Scheme 4. Suggested mechanism for the Michael addition of pyrrolidine to acrylonitrile.

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Apart from the structure of the reagents, the nature of the organic solvent is also important in enzyme catalysis. Hydrophobic solvents are favored because hydrophilic solvents can strip away the tightly bound water layer from the enzyme surface, which is required to maintain the catalytically active conformation of the enzyme.4 The partition coefficient (P) is the most widely used parameter for the measure of solvent polarity. (P) is defined as the ratio of the equilibrium concentrations of a solvent partitioned in a two-phase system consisting of n-octanol and water. A solvent with a (log P) value greater than 2 ensures high retention of the activity of the enzyme.21 The work of Riva and coworkers22 (Scheme 5) illustrates the effect of solvent hydrophilicity on the initial rate and conversion in CALB-catalyzed transesterification of vinyl acetate with cyclohexanol. The reaction was quantitative in all cases except DMF, which had a log P of −1.0, and the enzyme showed the highest activity in hexane, the solvent with the highest log P (Table 2).

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Scheme 5. CALB-catalyzed transesterification of vinyl acetate with cyclohexanol.

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Table 2. Effect of Solvent Hydrophilicity on Enzyme Activity
Solventlog PInitial Rate (μmol/min)Conversion (%)
5 h24 h
Hexane3.52.14100 
THF0.491.1795100
Acetonitrile−0.330.7685100
DMF−1.00

The optimum temperature range for CALB enzyme stability is 40–60 °C.23 For example, Zong and coworkers24 demonstrated that 50 °C was the optimum temperature for the acylation of 5-azacytidine with vinyl laurate in pyridine (Scheme 6).

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Scheme 6. CALB-catalyzed transesterification of 5-azacytidine with vinyl laurate.

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It must be pointed out that in most of the examples shown above, CALB was immobilized on a macroporous acrylic resin by physical adsorption (Novozyme 435®, from Novozymes A/S). Immobilization increases the thermal stability by stabilizing the tertiary structure of the protein, and facilitates the removal of the enzyme from the reaction mixture allowing repeated use.4, 25 Furthermore, adsorbing the enzyme on a solid support can increase the surface area of the enzyme by preventing it from aggregation in organic solvents, which in turn can enhance the catalytic activity.26

ENZYMES IN POLYMER CHEMISTRY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information

Polymer Synthesis

The use of enzymes for polymer synthesis has been reviewed in detail.27–31 The most important examples are polycondensation, oxidative polymerization, and ring opening polymerization. Examples are illustrated below.

Polycondensation

Polyesters have been effectively synthesized through lipase-catalyzed polycondensation of hydroxyacids or their esters32 (A-B type condensation) (Scheme 7) and dicarboxylic acids or their derivatives with glycols33 (AA-BB type condensation) (Scheme 8).

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Scheme 7. Lipase-catalyzed condensation polymerization of 11-hydroxyundecanoic acid.

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Scheme 8. Lipase-catalyzed polycondensation between bis(2,2,2-trichloroethyl) adipate and 1,4-butanediol.

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Oxidative Polymerization

Some oxidoreductases such as peroxidase and laccase have been employed for the oxidative polymerization of phenols and aniline derivatives yielding polyaromatic compounds.4 Among oxidoreductases, horseradish peroxidase (HRP) is the most widely used catalyst in oxidative polymerizations. The active site of the enzyme is an iron-protoporphyrin (Scheme 9). The reaction mechanism involves the oxidation of Fe+3 to an iron-oxo derivative at the expense of H2O2; followed by abstraction of an electron from an electron-rich substrate, for example, a phenol, to from a substrate radical, that is, a phenoxy radical (Scheme 9).4 The resulting radicals then react to form polymers via migration and subsequent recombination as shown below (Scheme 10).34

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Scheme 9. The catalytic cycle of HRP for a phenol substrate and the structure of HRP active site.

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Scheme 10. HRP-catalyzed polymerization of 4-ethylphenol.

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Ring Opening Polymerization

A common methodology in which enzymes are employed is the synthesis of polyesters by lipase-catalyzed ring opening polymerization (ROP) of lactones. For example, Svirkin et al.35 demonstrated ROP of a racemic mixture of α-methyl-β-propiolactone using lipase from Pseudomonas fluorescens (lipase PF). The enzyme showed stereoselectivity toward the (S)-enantiomer, and an (S)-enriched polymer with up to 75% (S) was formed (Scheme 11).

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Scheme 11. Lipase-catalyzed ROP of α-methyl-β-propiolactone.

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By analogy with other lipase-catalyzed reactions, Uyama et al.36 suggested that the reaction mechanism (Scheme 12) involved the ring opening of lactone by the nucleophilic attack of serine residue in the active site of the enzyme to give the acyl-enzyme intermediate (enzyme activated monomer), followed by deacylation of the acyl-enzyme by nucleophiles such as water or alcohol and by the terminal hydroxyl group of a growing polyester in the initiation and propagation steps, respectively.

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Scheme 12. Mechanism suggested for lipase-catalyzed ring opening polymerization.

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ROP is widely employed for the preparation of polyester macromonomers using functionalized initiators and/or terminators. To prepare methacrylate-terminated polyesters, Kobayashi and coworkers37 polymerized 12-dodecanolide (13-membered lactone) (DDL) using 2-hydroxyethyl methacrylate (HEMA) as the alcohol initiator and vinyl methacrylate as the terminating agent38 in the presence of CALB and lipase PF, respectively; and conversions were nearly quantitative. However, a recent study reported by Martinelle and coworkers39 revealed that HEMA-initiated, CALB-catalyzed ROP of ω-pentadecalactone (PDL), and ε-caprolactone (CL) resulted in a mixture of various polyester methacrylate structures. The enzyme also catalyzed the cleavage of the ester bond in HEMA end groups in preformed polyesters, therefore limited the use of initiators with ester functionalities in ROP for the preparation of well-defined polyester macromonomers. Telechelic polyesters with carboxylic acid groups at both chain-ends were also prepared via lipase PF, which catalyzed ROP of DDL using divinyl sebacate that acted as the coupling agent for the hydroxyl ends of propagating poly(DDL) chains.40 Cordova et al.41 polymerized CL in the presence of unsaturated alcohols, unsaturated fatty acids and esters, vinyl esters and phenolic compounds using CALB to obtain the corresponding functionalized polyesters. CALB-catalyzed regioselective acylation was exploited to prepare methyl 6-O-poly(ε-caprolactone)-β-D-glucopyranoside42 and poly(ε-caprolactone) monosubstituted dendrimer43 using methyl β-D-glucopyranoside and a hexahydroxyl-functional dendrimer, respectively, as polymerization initiators. Thiol-functionalized polycaprolactone (PCL) with high thiol chain-end functionality was also prepared with the same enzyme using 2-mercaptoethanol as the initiator due to the chemoselectivity of CALB toward alcohols over thiols in transacylation.44 The same strategy was extended to prepare α,ω-functionalized polypentadecalactones in bulk with dithiol, diacrylate, or thiol-acrylate end groups using (a) 6-mercapto-1-hexanol as initiator and 11-mercapto-1-undecanoic acid or vinyl acrylate as terminator or (b) water as initiator and ethylene glycol diacrylate as terminator.45 Epoxy-functionalized PCL was synthesized by glycidol-initiated ROP in the presence of CALB.46 MALDI-ToF MS revealed that there was only one distribution corresponding to linear PCL with glycidol end groups and that the epoxy ring of the glycidol remained intact.

Enzyme-Catalyzed Post-Polymerization Functionalization of Polymers

Our literature search revealed that very little had been done in the area of post-polymerization functionalization catalyzed by enzymes, and most examples were hampered by low efficiency.

Natural Polymers

Most of the publications in enzymatic polymer modification deal with water soluble, natural polymers, particularly polysaccharides. Hydroxyethylcellulose (HEC) was transgalactosylated using lactose as the donor in the presence of β-galactosidase from Aspergillus oryzae in sodium acetate buffer.47 Although the number of hydroxyl sites within each HEC unit available for transgalactosylation was 3, the maximum degree of substitution obtained by this method was only 0.033. The same natural polymer, HEC, has been modified with succinic anhydride, vinyl stearate, vinyl acetate, and vinyl acrylate through transesterification in N,N-dimethylacetamide (DMAc) solvent using lipase from Pseudomonas cepacia.48 The use of subtilisin Carlsberg in anhydrous pyridine has also been reported for the functionalization of the HEC with vinyl propionate and vinyl acrylate.49 However, in all HEC modification reactions, low conversions have been observed. Tyrosinase was used to graft various phenolic substrates onto chitosan.50 It was proposed that the enzyme converted phenols into reactive o-quinone intermediates, which subsequently underwent nonenzymatic reactions with the nucleophilic amine groups of chitosan. In spite of the low conversions, these modifications resulted in dramatic changes in polymer properties. Understanding the mechanism of the reactions remained a challenge. A thin film of amylose, an organic solvent-insoluble polysaccharide, was regioselectively acylated with fatty acid esters in isooctane using subtilisin Carlsberg enzyme, which was made soluble in organic medium via surfactant ion-pairing.51 However, the insolubility of the polymer in the organic medium led to reduced reaction rates; and the degree of substitution per glucose moiety in the polymer was found to be 0.185.52 β-cyclodextrin and HEC were also acylated using the same methodology.52 Bakers' yeast hexokinase was used for the phosphorylation of cellulose in the presence of phosphoryl donor, ATP.53 Phosphorylation of 0.03% of the glycopyranose units in the cellulose resulted in improved dyeability and flame resistance.

Synthetic Polymers

As mentioned earlier, the number of examples involving post-polymerization enzymatic functionalization of synthetic polymers is also rather limited. Jarvie et al.54 showed that CALB was able to catalyze the epoxidation of polybutadiene selectively in the presence of hydrogen peroxide and catalytic amount of acetic acid. Approximately 60% of the 1,4-cis and trans double bonds in the backbone was epoxidized, whereas the pendant vinyl groups remained intact. The inability to reach higher conversions was attributed to the conformational changes in the partially epoxidized polymer. The pendant nitrile groups in polyacrylonitrile fibers were converted to the corresponding amides by a nitrile hydratase enzyme with 16% conversion.55 Poly(acrylic acid) backbone was modified through esterification with polyols in bulk with nearly 100% regioselectivity using CALB.56 When glycerol was used as the polyol, conversions up to 32% were reached as determined by 1H NMR spectroscopy. It was also found in the same study that CALB did not catalyze intermolecular reactions between the pendant free hydroxyl groups of the polyol and the carboxylic acid groups of another polyacrylic acid; thus, no formation of crosslinks between chains was observed. Vinyl acetate was grafted onto poly(styrene-co-(p-vinylphenylethanol)) having about 45% secondary hydroxyl content by transesterification in toluene using CALB.57 The maximum conversion was 75%, and changing the reaction parameters did not increase the conversion significantly. Similarly, when poly(styrene-co-(4-vinyl-benzyl alcohol)) containing 10% primary hydroxyl functionality was used, vinyl acetate grafting was 95%.58 The same researchers inverted the structural units, namely incorporated the ester functionality into the polymer [i.e., poly(styrene-co-methyl-2-(4-styryl) acetate)] and reacted the polymer with an alcohol (e.g., benzyl alcohol).59 They observed that there was no reaction at all. This was overcome by introducing a spacer in the ester-functionalized polymer, and up to 78% conversion was reached. Lipase PF catalyzed acylation of poly[N-(2-hydroxypropyl)-11-methacryloylaminoundecanamide-co-styrene], a comb-like polymer, and the corresponding monomer with vinyl acetate, phenyl acetate, 4-fluorophenyl acetate, and phenyl stearate was reported by Pavel and Ritter.60 It was found that the copolymer could be acylated with about 40% conversion when phenyl acetate was used and that the reactivity of the monomer was higher than that of the copolymer indicating the effect of steric hindrance on reaction kinetics.

Poly(ethylene glycol) (PEG) was functionalized by galactose in water through the use of lactose as the donor and β-galactosidase as the enzyme.61 Based on NMR analysis, the conversion was about 30–50%. Fatty acid functionalities were also added onto amine terminated-PEG through CALB-catalyzed aminolysis in bulk using methyl palmitate, but conversion data were not reported.62 Gross and coworkers63 reported the synthesis of organosilicon carbohydrate macromers by CALB-catalyzed esterification. Diacid end-blocked siloxanes were reacted with α,β-ethyl glucoside under vacuum in bulk. Esterification occurred with high regioselectivity (>98%) at the primary hydroxyl (C6) of the glucoside; and the extent of esterification was almost 99% as determined by electrospray ionization mass spectrometry (ESI MS).

Against this background, we started to investigate the use of enzymes for precision functionalization of polymers.

Precision Polymer Functionalization via Enzymatic Catalysis

We started to investigate polymer functionalization using enzymatic catalysis based on the desire of biomaterial functionalization using “green” polymer chemistry. This method would not leave undesirable catalyst residues in materials intended for in vivo applications. Because most of the polymer-related use of enzymes seemed to be based on trial and error, we started with model reactions to get a better understanding of the reaction mechanism.

Model Reactions
Transesterification.

2-phenyl-1-propanol (2PPOH) is a model compound for OH-functionalized PIB obtained by living carbocationic polymerization initiated by the α-methylstyrene epoxide (α-MSE)/TiCl4 initiator system.64 The transesterification of vinyl acetate with 2PPOH using bis(dibutylchloro-tin(IV)) oxide in THF at 50 °C for 12 h remained incomplete, in contrast to full conversion with CALB in hexane in 2 h (Scheme 13).65

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Scheme 13. Comparison of catalytic activities of CALB and distannoxane in transesterification of vinyl acetate with 2PPOH.

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The transesterification of vinyl methacrylate (VMA) with ethylene glycol (EG) (Scheme 14), a model for poly(ethylene glycol) (PEG) was carried out in THF because PEG is insoluble in hexane. About 1.5 mol eq of VMA per OH groups was added to the EG, and the progress of the reaction was monitored by TLC.66 Six hours after the start of the reaction, in addition to the ethylene glycol spot at Rf = 0.15, a new spot was detected at Rf = 0.60. No further change was observed until t = 24 h when 1.5 more equivalents of VMA was added. The TLCs taken up to 12 h reaction time showed only two spots at Rf = 0.15 and 0.6. The TLC taken after 18 h reaction time showed two spots at Rf = 0.60 and 0.85, while the ethylene glycol spot at Rf = 0.15 disappeared. After 36 h reaction time, the TLC showed a single spot at Rf = 0.85. Quantitative conversion was confirmed by 1H NMR.66 Based on the TLC monitoring, the enzyme-catalyzed transesterification with ethylene glycol progressed in a consecutive fashion, first yielding the monofunctionalized ester (Rf = 0.6), followed by the difunctionalized product (Rf = 0.85).

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Scheme 14. CALB-catalyzed transesterification of vinyl methacrylate with ethylene glycol.

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Thiol-functionalized polymers have many applications including stabilization of gold nanoparticles for drug delivery67, 68 and sensors for heavy metal ions.69, 70 The model 11-mercapto-1-undecanol (MEUD) was reacted with a slight excess of divinyl adipate (Scheme 15), yielding quantitatively the monofunctionalized product, as shown in Figure 2. The vinyl ester is available to attach hydroxyl-functionalized polymers to this intermediate using CALB. Thus, the entire synthesis will take two steps, without the need of protecting the thiol group. This also demonstrates the attractiveness of enzyme-catalyzed polymer functionalization.

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Figure 2. 1H NMR of transesterification product of 11-mecapto-1-undecanol to divinyl adipate (solvent: CDCl3).

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Scheme 15. Synthesis of 11-mercaptoundecyl vinyl adipate using CALB.

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Michael Addition.

We have successfully used thymine-functionalized polystyrene for chemisorption of proteins and drugs via hydrogen bonding71, 72; thus, we were interested in thymine-functionalization via enzymatic catalysis. We carried out model reactions of Michael addition of thymine to both vinyl acrylate (VA) (Scheme 16) and vinyl methacrylate (VMA). The conversion with VMA was only 63%, whereas quantitative addition was obtained with VA as determined by 1H NMR (Fig. 3).

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Figure 3. 1H NMR of the Michael addition product of thymine to vinyl acrylate (solvent: DMSO-d6).

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Scheme 16. Michael addition of thymine to vinyl acrylate.

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It was somewhat surprising that the presence of a single methyl group connected to the α-carbon hindered the reaction, but we found this effect in other reactions. CALB-catalyzed transesterification of ethylene glycol (EG) with the vinyl ester of thymine (TVMA) (Scheme 17) prepared by Michael addition of thymine to VMA gave a difunctional product, but only with 43% conversion (Fig. 4).

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Figure 4. 1H NMR of the transesterification product of EG with TVMA (solvent: DMSO-d6).

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Scheme 17. Transesterification of EG with TVMA.

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Subsequently, we synthesized a compound with one acrylate and one methacrylate terminus by reacting hydroxyethyl acrylate with vinyl methacrylate (Scheme 18).

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Scheme 18. Transesterification of HEA with VMA.

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Michael addition of 2,2′-(ethylenedioxy)-bis(ethylamine) to this compound proceeded exclusively and quantitatively with the acrylate terminus (Scheme 19) as shown in Figure 5.

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Figure 5. 1H NMR spectrum of the Michael addition product of HEA-VMA with 2,2′-(ethylenedioxy)-bis(ethylamine) (solvent: DMSO-d6).

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Scheme 19. Michael addition of HEA-VMA with 2,2′-(ethylenedioxy)-bis(ethylamine).

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Following the model reactions, we turned our attention to polymer functionalization.73

Functionalization of Polyisobutylenes

PIB is a biocompatible nonpolar polymer, used in chewing gums and drug-eluting stent coatings.74–77 We reported the quantitative functionalization of PIB-OHs with different chain end-structures (Scheme 20) in hexane.78

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Scheme 20. Enzymatic functionalization of PIB-OH (Mn = 5200 g/mol, Mw/Mn = 1.09) and Glissopal-OH (Mn = 3600 g/mol, Mw/Mn = 1.34).

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Recently, we succeeded with functionalization in bulk! The immobilized enzyme was mixed with liquid PIB-OH, made from PIB-Cl as reported79 (Scheme 21). Three equivalents of VMA were added to the mixture and after 12 h, the reaction was complete. The enzyme was filtered, and excess VMA was removed by vacuum, yielding the clean product. The 1H NMR is shown in Figure 6. The methylene protons adjacent to the hydroxyl group at δ = 3.29–3.52 ppm shifted downfield on methacrylation as expected, and the integration ratio of these protons to the vinylidene (δ = 5.55 and 6.10 ppm) and methyl (δ = 1.95 ppm) protons of the newly formed methacrylate-end indicated quantitative functionalization. The same reaction was also successful using Glissopal-OH (Mn = 3600 g/mol, Mw/Mn = 1.34), which was obtained from commercially available Glissopal®2300 (BASF).

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Figure 6. 1H NMR spectrum of methacrylation product of liquid PIB-OH in bulk (Mn = 1500 g/mol, Mw/Mn = 1.29) (solvent: CDCl3).

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Scheme 21. Preparation of hydroxyl-functionalized PIB.

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Functionalization of Siloxanes

Commercially available liquid poly(dimethyl siloxane)s (PDMS) (silanol-terminated PDMS, Mn = 3200 g/mol; Aldrich, monocarbinol-terminated PDMS, Mn = 5000 g/mol; Gelest, and dicarbinol-terminated PDMS, Mn = 4500 g/mol; Gelest) were quantitatively functionalized with 1.5 eq. of vinyl methacrylate in bulk within 2 h, yielding the products shown in Scheme 22. Figure 7 shows the 1H NMR of a representative example.

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Figure 7. 1H NMR of the methacrylation product of PDMS monocarbinol (Mn = 5000 g/mol, Gelest) in bulk (solvent: CDCl3).

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Scheme 22. Methacrylated PDMS products.

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Functionalization of liquid polymers under solvent-less conditions is a very attractive feature.

Functionalization of Poly(ethylene glycols) PEGs

The nature of the active site pocket of CALB is hydrophobic,13 and the enzyme was very effective in methacrylation of hydrophobic polymers as discussed above. We also wanted to see if we can enzymatically functionalize a hydrophilic polymer. For this purpose, we choose PEG, which is one of the most widely used polymers in biomedical applications.80 We reported the quantitative methacrylation of a series of PEGs using CALB (Scheme 23).661H NMR and MALDI-ToF MS verified the expected structure and very clean products for all PEG samples listed in Table 3.66

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Scheme 23. CALB-catalyzed methacrylation of PEG.

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Table 3. Poly(ethylene glycol)s Used in the Enzymatic Transesterification of VMA
Mna (g/mol)Mn (g/mol) (1H NMR)Mw/Mn
  • a

    Nominal values given by the supplier.

1,0009501.16 (1.06a)
10,1008,0001.05a
2,0002,0501.91
4,6005,9502.35
10,00012,8002.11

One of the biomedical applications in which PEG is employed is as a carrying agent in drug delivery.81 Scheme 24 and Figure 8 demonstrate the attachment of a single PEG to a trifunctional acrylate, via enzyme-catalyzed Michael addition. The two remaining acrylate groups are available for the attachment of therapeutic and imaging agents.

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Figure 8. 1H NMR spectrum of the Michael addition product of ω-amino terminated poly(ethylene glycol) methyl ether to 1,3,5-triacryloylhexahydro-1,3,5-triazine (solvent: DMSO-d6).

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Scheme 24. CALB-catalyzed Michael addition of ω-amino terminated poly(ethylene glycol) methyl ether (Mn = 2000 g/mol, Mw/Mn = 1.05) to 1,3,5-Triacryloylhexahydro-1,3,5-triazine.

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We also synthesized ditelechelic PEG-thymine by Amano lipase M (Aldrich)-catalyzed Michael addition of thymine to ditelechelic PEG-acrylate, which was prepared via transesterification of vinyl acrylate with HO[BOND]PEG[BOND]OH (Mn = 2000 g/mol; Mw/Mn = 1.91, Aldrich) (Scheme 25). As 1H NMR shows (Fig. 9), the acrylate protons at δ: 5.9–6.4 ppm disappeared and the methylene protons (b) next to the ester bond shifted upfield after the reaction. The integration ratios of the thymine protons and the methylene protons (b) next to the ester bond were in good agreement with the expected structure. This construct can self-assemble via H-bonding, and drugs such as Ciprofloxacine can be hydrogen bonded to the thymine residue.72

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Figure 9. 1H NMR of the product of Amano-lipase catalyzed Michael addition of thymine to ditelechelic PEG-acrylate (solvent: DMSO-d6).

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Scheme 25. Preparation of thymine-functionalized PEG.

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Regioselectivity in Enzymatic Polymer Functionalization

Methacrylation of PIB-OH obtained from α-MSE/TiCl4 initiated polymerization of isobutylene did not proceed even though the model reaction was quantitative as discussed in the model reaction section. First, we suspected interference from the Cl end group. However, the removal of the tertiary chloride chain end by dehydrohlorination using t-BuOK did not result in any change in polymer reactivity toward the enzyme. Subsequently, starting with the PIB-OH obtained from α-MSE/TiCl4 (Mn = 7200; Mw/Mn = 1.04), we prepared an asymmetric α,ω-hydroxyl-functionalized PIB via the method shown in Scheme 21. The asymmetric ditelechelic PIB with two primary OH groups was reacted with VMA in the presence of CALB (Scheme 26). 1H NMR revealed that only the primary hydroxyl formed by the derivatization of the chloro chain-end reacted (Fig. 10). The methylene protons adjacent to the hydroxyl group shifted downfield on methacrylation and new peaks corresponding to the methacrylate end-group were observed at the expected positions, whereas the methylene protons adjacent to hydroxyl group at the phenyl end remained intact.

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Figure 10. 1H NMR of the product of transesterification of VMA with α,ω-primary-hydroxy telechelic PIB (solvent: CDCl3).

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Scheme 26. Regioselective enzymatic methacrylation of α,ω-hydroxyl-functionalized PIB.

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Therefore, we concluded that most likely after the formation of the acyl-enzyme complex by the nucleophilic attack of serine to the carbonyl group of VMA, the more sterically hindered hydroxyl group could not coordinate with the carbonyl of the acyl-enzyme complex because of the bulky polymer chain preventing the most suitable conformation (Scheme 27). In contrast, the 2PPOH model, having a H in place of the PIB chain (Scheme 13), had enough space for coordination with the acyl-enzyme complex, and thus, quantitative methacrylation could be obtained.

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Scheme 27. Unsuccessful methacrylation of PIB-OH prepared from α-MSE/TiCl4 initiator system.

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The effect of steric hinderance was further demonstrated using primary hydroxyl-functionalized polystyrene. We observed that even after 48 h, there was no progress in methacrylation of PS-OH, which was prepared by end-capping of poly(styryl)lithium with ethylene oxide (see Scheme 28). In contrast, PS-OH synthesized by the general functionalization methodology reported by Quirk et al.82 via end-capping of poly(styryl)lithium with chlorodimethylsilane followed by hydrosilation with allyl alcohol gave complete conversion in 48 h (Fig. 11).

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Figure 11. 1H NMR spectrum of the methacrylation product of PS-OH prepared by end-capping of PSLi with (CH3)2SiClH followed by hydrosilation with allyl alcohol (solvent: CDCl3).

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Scheme 28. Unsuccessful transesterification of vinyl methacrylate with PS-OH prepared by ethylene oxide end-capping (Mn = 2100 g/mol, Mw/Mn = 1.07) (top); quantitative transesterification of vinyl methacrylate with PS-OH prepared by end-capping with (CH3)2SiClH followed by hydrosilation with allyl alcohol (Mn = 2600 g/mol, Mw/Mn = 1.06) (bottom).

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Introduction of the [Si(CH3)2[BOND]CH2] spacer between the phenyl ring of the last styrene unit and the hydroxyl chain-end rendered PS-OH reactive for enzymatic methacrylation.

SYNOPSIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information

We herein showed an alternative, quantitative, cost affective, and environmentally benign way of producing functionalized polymers for biomedical and other applications via enzyme-catalyzed transesterification and Michael addition. Successful functionalization of liquid polymers under solvent-less conditions makes this approach even more attractive. Our work focuses on drug delivery applications.

The use of enzymes as catalysts in polymer science is a powerful methodology for the preparation of novel polymeric structures, which are either difficult or impossible to synthesize by classical synthetic pathways. A better understanding of the way these mysterious catalysts of nature work would allow us to design new materials with unique properties. We are investigating the mechanism of these reactions in our laboratory.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information

This work is supported by the National Science Foundation under the grants DMR-0509687 and #0804878. The authors thank E. Wichman and R. P. Quirk for providing PS-OH samples. We wish to thank the Ohio Board of Regents and the National Science Foundation (CHE-0341701 and DMR-0414599) for funds used to purchase the NMR instrument used in this work.

REFERENCES AND NOTES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information

Biographical Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information
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JUDIT E. PUSKAS

Dr. Puskas received a PhD degree in plastics and rubber technology in 1985, in the framework of collaboration between the National Science Foundation of the USA and the Hungarian Academy of Sciences. Her advisors were Professors Ferenc Tüdös and Tibor Kelen of Hungary, and Professor Joseph P. Kennedy at the University of Akron, Ohio, USA. She started her academic career in 1996. Before that she was involved in polymer research and development in the microelectronic, paint, and rubber industries. Her present interests include green polymer chemistry, biomimetic processes and biomaterials, living/controlled polymerizations, polymerization mechanisms and kinetics, thermoplastic elastomers and polymer structure/property relationships, and probing the polymer-bio interface. She is one of the editors of the new Interdisciplinary Reviews in Nanomedicine and NanoBiotechnology WIRE, published by Wiley-Blackwell, and a member of the Advisory Board, of the European Polymer Journal. Until July of 2008, she was one of the two regional Editors with the highest citation index. Puskas has been published in more than 300 publications, including technical reports, is an inventor or co-inventor of 20 U.S. patents and applications, and has been Chair or organizer of a number of international conferences. She is the recipient of several awards, including the 1999 PEO (Professional Engineers of Ontario, Canada) Medal in Research and Development, a 2000 Premier's Research Excellence Award, the 2004 Mercator Professorship Award from the DFG (Deutschen Forschungsgemeinschaft, German Research Foundation), and the 2008 “Chemistry of Thermoplastic Elastomers” Award of the Rubber Division of the American Chemical Society.

Puskas was Group Leader of Butyl Technology in the Rubber Division of Bayer Inc. before she left industry. From 1998 to 2003, she held the Bayer/NSERC (Natural Science and Engineering Research Council of Canada) Industrial Research Chair in Elastomer technology, and was the Director of the Macromolecular Engineering Research Centre at the University of Western Ontario in Canada from 1996 to 2004. In August of 2004, she joined the Faculty in the Department of Polymer Science of the College of Polymer Science and Polymer Engineering of the University of Akron, where she held the LANXESS (previously Bayer) Industrial Chair until 2008. She has been awarded her third NSF grant in 2008. As a coinventor of the polymer used on the Taxus® coronary stent, she helped the University of Akron to generate more than $5 million in license fees.

Biographical Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information
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MUSTAFA Y. SEN

Mustafa Y. Sen is a PhD student in the Department of Polymer Science, University of Akron. He obtained his BS degree in Chemistry at Bogazici University, Turkey in 2002 and MS degree in Polymer Science at University of Akron in 2005 under the supervision of Dr. Roderic P. Quirk. He joined the Puskas group in December of 2005. He is the first student working on the functionalization of polymers using enzymatic catalysis, a new research direction in the Puskas group. His research interests include the enzymatic functionalization of biomaterials and getting a better fundamental understanding of enzyme catalysis.

Biographical Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENZYMES IN POLYMER CHEMISTRY
  5. SYNOPSIS
  6. Acknowledgements
  7. REFERENCES AND NOTES
  8. Biographical Information
  9. Biographical Information
  10. Biographical Information
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KWANG SU SEO

Kwang Su Seo is a PhD student in the Department of Polymer Science, University of Akron. He obtained his BS degree in Polymer Science and Technology in 2002 and MS degree in Polymer Nanotechnology in 2005 under the supervision of Dr. Gilson Khang, Dr. Hai Bang Lee, and Dr. Moon Suk Kim at Chonbuk National University, Korea. He joined the Puskas group in the fall of 2007. His research interests include the enzyme catalyzed synthesis of star and dendritic polymers for preparing multivalent drug delivery carriers, in collaboration with the University of Michigan.