bk-2013-1144.ch007

Chapter 7

Downloaded by TROY UNIV on October 16, 2014 | http://pubs.acs.org Publication Date (Web): November 22, 2013 | doi: 10.1021/bk-2013-1144.ch007

Green Polymer Chemistry: Enzymatic Functionalization of Poly(ethylene glycol)s Under Solventless Conditions Judit E. Puskas,*,1,2,3 Kwang Su Seo,2 Marcela Castaño,2 Madalis Casiano,3 and Chrys Wesdemiotis2,3 1Department

of Chemical and Biomolecular Engineering, The University of Akron, Akron, Ohio 44325, U.S.A. 2Department of Polymer Science, The University of Akron, Akron, Ohio 44325, U.S.A. 3Department of Chemistry, The University of Akron, Akron, Ohio 44325, U.S.A. *E-mail: [email protected]

Enzymes as biocatalysts have been contributing to “Green Polymer Chemistry”, providing an alternative to conventional chemical catalysis for polymerization and polymer functionalization. Specifically, enzyme-catalyzed polymer functionalization carried out under solventless conditions is a great advancement in the design of a green process for biomedical applications, where the toxicity of solvents and catalyst residues need to be considered. In this paper, we present quantitative enzyme-catalyzed transesterification of vinyl esters with poly(ethylene glycol)s (PEG)s. Specifically, vinyl methacrylate, vinyl acrylate and vinyl crotonate were transesterified with PEGs in the presence of Candida antarctica lipase B (CALB; Novozym® 435) at 50 °C under solventless conditions. 1H and 13C NMR spectroscopy and MALDI-ToF mass spectrometry verified the structure and the purity of the functionalized polymers.

© 2013 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Introduction In tune with the globally increasing interest in “Green or Greener” chemistry, our group has been exploring the power of enzyme catalysis in the functionalization of synthetic polymers. Enzymatic catalysis in organic synthesis has emerged as an attractive “green chemistry” alternative to conventional chemical catalysis. In the past two decades, enzymatic catalysis has been applied to polymer synthesis (1–5) and functionalization (6–11) with several advantages, including high efficiency, recyclability, the ability to operate under mild conditions, and environmental friendliness (12). All enzymes are classified into six main groups according to the International Union of Biochemistry and Molecular Biology (Figure 1) (7, 12). Today about 3000 enzymes are commercially available and some of them are mutated for industrial applications. Generally, oxidoreductases, hydrolases, and isomerases are relatively stable, and the most widely used catalysts in biotransformations. In contrast, lyases and ligases are present in lesser amounts in living cells and are less stable for isolation or separation from living organisms.

Figure 1. Classification of enzymes. Reproduced with permission from ref. (7). Copyright 2011 Elsevier.

Lipases belong to the group of hydrolyses and are the most popular biocatalysts. They are widely used in esterification, transesterification, aminolysis, and Michael addition reactions in organic solvents (12, 13). The most useful lipases for organic synthesis are: porcine pancreatic lipase (PPL), lipase from Pseudomoanas cepacia (Amano lipase PS, PCL), lipase from Candida rugosa (CRL), and lipase B from Candida antarctica (CALB) (13). CALB is one of the most effective catalysts for transesterification. CALB contains 317 amino acids and the catalytic triad is Ser105- His224-Asp187 (14). It has a larger pocket above the Asp-His-Ser triad and a medium-sized pocket below it (Figure 2.) (9, 15, 16). The top pocket is the “carbonyl” pocket, and the bottom is the “hydroxyl” pocket, represented by different shading in Figure 2. 82 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 2. (Left) 3D structure of CALB [Image of PBD ID:1TCB from ref. (15) created with Polyview 3D] and (right) schematic representation of the the catalytic triad of CALB.

Since we will discuss PEG functionalization via transesterification, this reaction will be discussed in more detail.

CALB-Catalyzed Transesterification Transesterification reactions are generally reversible. In order to shift the equilibrium towards the product, the nucleophilicity of the leaving group of the acyl donor should be reduced by the introduction of electron-withdrawing groups (e.g., trihaloesters, enol esters, oxime esters, anhydrides, etc), described in Scheme 1 (17).

Scheme 1. Transesterification of esters with alcohols. The use of enol esters (17, 18) such as vinyl or isopropenyl esters appears to be the most useful since it liberates unstable enols as by-products which rapidly tautomerize to give the corresponding aldehydes or ketones. Therefore, the reaction becomes irreversible. Acetaldehyde, which forms during the reactions with vinyl esters, is known to inactivate the lipases from Candida rugosa and Geotrichum candidum by forming a Schiff’s base with the lysine residues of the protein; however most lipases, including CALB, tolerate the liberated acetaldehyde (18). Comparison of the catalytic activities of CALB and distannoxane, a conventional tin-based catalyst revealed that the transesterification of vinyl acetate with 2-phenyl-1-propanol catalyzed by CALB was complete in 2 hours, while the traditional tin-based catalyst yielded 95% conversion in 12 hours (Figure 3) (7). 83 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 3. Transesterification of vinyl acetate: comparison of CALB and a tin-based catalyst. Reproduced with permission from ref. (7). Copyright 2011 Elsevier.

The catalytic cycle of the CALB-mediated transesterification shown in Figure 3 can be visualized based on the mechanism described in the literature (8, 9, 13, 15). The different shadings in our rendition in Figure 4 represent the carbonyl and hydroxyl pockets of the enzyme. First, the nucleophilic serine (Ser105) residue interacts with the carbonyl group of the vinyl acetate, forming a tetrahedral intermediate which is stabilized by the oxyanion hole of the enzyme via three hydrogen bonds: one from glutamine (Gln106) and two from threonine (Thr40) units. In the second step, the ester bond is cleaved to form the acyl-enzyme complex (AEC) and the first product, vinyl alcohol. Vinyl alcohol will immediately tautomerize to acetaldehyde, rendering the reaction irreversible. In the third step, the alcohol reacts with the acyl-enzyme complex to form a second tetrahedral intermediate which is again stabilized by the oxyanion hole. In the last step, the enzyme is deacylated to form the desired product. The nucleophilic attack by the Ser105 is mediated by the His224-Asp187 pair.

84 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 4. Illustration of the mechanism of CALB-catalyzed transesterification of vinyl acetate with 2-phenylpropane-1-ol. The different shading represents the two enzyme pockets. Enzymes in the Synthesis of Telechelic Polymers Before our group had started working on the end-functionalization of preformed synthetic symmetric or asymmetric telechelic polymers, enzymatic catalysis has not been extensively utilized in this area despite the advantages enzymes offer. Telechelic carboxylic acid functionalized PDMSs were reacted with α,β-ethylglucoside at 70 °C under vacuum for 34 hours in the presence of CALB, but the product was a mixture of mono- and difunctional esters (19). Recently the synthesis of telechelic methacrylate-functionalized oligoesters by the CALB-catalyzed polycondensation of ethylene glycol, hydroxyethyl methacrylate and divinyl adipate (DVA) was reported (20). However, 5-9% of the product was not difunctional methacrylate. The first examples of quantitative functionalization of telechelic polymers using CALB-catalyzed reactions with and without organic solvents have been reported by our group (7–11). The primary hydroxyl groups 85 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


of hydroxyl-functionalized polyisobutylenes (PIBs) were quantitatively methacrylated by transesterification of vinyl methacrylate in the presence of CALB within 24 hours in hexane and 2 hours in bulk, respectively (Scheme 2) (8, 9).

Scheme 2. CALB-catalyzed transesterification of vinyl methacrylate with PIBOHs. Commercially available polydimethylsiloxanes (PDMS), PDMSmonocarbinol and PDMS-dicarbinols were also methacrylated with vinyl methacrylate under solventless conditions within 2 hours in the presence of CALB (8, 9). The methacrylation and acrylation of PEGs with various molecular weights and molecular weight distributions in THF were quantitative in 24 hours. Against this background, we investigated the transesterification of vinyl methacrylate, vinyl acrylate and vinyl crotonate with PEGs under solventless conditions

Enzymatic Functionalization of Poly(ethylene glycol)s under Solventless Conditions Transesterification of Vinyl Acrylate and Vinyl Methacrylate with PEG Solid low molecular weight PEGs became liquid at 50 °C and were quantitatively functionalized by transesterification. When reacted with vinyl methacrylate (VMA) in the presence of CALB under solventless conditions, the reactions were complete within 4 hours (Scheme 3).

Scheme 3. Enzymatic transesterification of VMA with PEG (Mn = 1000 g/mol; Mw/Mn = 1.08) in the presence of CALB. [VMA] = 8.32 mol/L, [PEG] = 0.83 mol/L; [CALB] = 5.0 × 10-4 mol/L. In the 1H NMR spectrum of Figure 5, the hydroxyl proton signals at δ=4.55 ppm (a, not shown) from the HO-PEG-OH disappeared and the peak corresponding to the methylene protons adjacent to the hydroxyl groups shifted downfield from δ=3.50 to δ=4.22 ppm (b) after the reaction. The new peaks 86 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


corresponding to the vinyl [δ=6.07 ppm (e) and δ=5.65 ppm (e′)] and methyl [δ=1.73 ppm (f)] protons of the methacrylate group were observed at the expected positions. The 13C NMR spectrum of the methacrylation product also confirmed the structure of the polymer. The 13C NMR spectrum showed that the carbons connected to the hydroxyl groups at δ=60.13 ppm in the HO-PEG-OH shifted downfield to δ=64.02 ppm (B) and the carbon signals associated with the methacrylate group at δ = 17.95 ppm (G) δ = 136.17 ppm (F), δ = 126.20 ppm (E) and δ = 166.68 ppm (H) appeared after 4 hours reaction.

Figure 5. NMR spectra of the methacrylation product of PEG (Mn = 1000 g/mol; Mw/Mn = 1.08): (top) 1H NMR spectrum and (bottom) 13C NMR spectrum (solvent: DMSO-d6). The MALDI-ToF mass spectrum is shown in Figure 6. The peak at m/z 1101.61 corresponds to the sodium complex of the 21-mer of PEG dimethacrylate. The calculated monoisotopic mass for this ion is 1101.70 Da [21 × 44.03 (C2H4O repeat unit) + 154.08 (C8H10O3 end groups) + 22.99 (Na+)]. Within the series, the peaks were separated by 44 Da, corresponding to an ethylene glycol repeating units. The minor distribution of peaks which differ from the main series by 16 m/z units (see bottom of Figure 6) was assigned to the K+ complex of PEG dimethacrylate - it is known that K+ contamination occurs during sample preparation (21). Therefore, MALDI-ToF MS analysis confirmed that the conversion of HO-PEG-OH to PEG dimethacrylate was quantitative within 4 hours of reaction time under solventless conditions. PEG with 2000 g/mol was also enzymatically methacrylated under the same conditions as above, yielding a very pure product. 87 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 6. MALDI-ToF mass spectrum of PEG dimethacrylate (Mn=1000 g/mol; Mw/Mn = 1.08).

The same reactions were also carried out with vinyl acrylate, also yielding exceptionally pure products. Figure 7 shows the 1H NMR spectrum of the PEG diacrylate. The peak corresponding to the methylene protons next to hydroxyl group shifted downfield from δ=3.50 to δ=4.42 ppm (b) after 4 hours. New resonances attributed to the vinylidene [δ=6.20 ppm (e)] and vinyl [δ=6.32 88 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


ppm (f) and δ=5.97 ppm (e′)] protons appeared with the integration ratios of (b):(e):(f):(e′) as 2:1:1:1. The 13C NMR spectrum of the transesterification product also confirmed the structure of the PEG diacrylate. The carbons connected to the hydroxyl groups in the starting material at δ=60.13 ppm shifted downfield to δ=63.41 ppm (B) and the carbon signals associated with the acrylate group at δ = 166.02 ppm (H), δ = 131.86 ppm (F) and δ = 128.55 ppm (E) appeared after the acrylation.

Figure 7. NMR spectra of the acrylation product of PEG (Mn = 1000 g/mol; Mw/Mn = 1.08): (top) 1H NMR spectrum and (bottom) 13C NMR spectrum (solvent: DMSO-d6).

Figure 8 shows MALDI-ToF mass spectrum of the transesterification product after 4 hours of reaction. The major distribution corresponds to PEG diacrylate. In the expanded spectrum, the representative peak at m/z 1117.65 corresponds to the sodium complex of the 22-mer of PEG diacrylate; its calculated monoisotopic mass is [22 × 44.03 (C2H4O repeat unit) + 126.11 (C6H6O3 end groups) + 22.99 (Na+)] 1117.73 Da. The peak at m/z 1105.65, which belongs to a minor distribution differing from the main series by 12 m/z units, corresponds to α-acetate, ω-acrylate functionalized PEG. The calculated monoisotopic mass for its 22-mer is 1105.75 Da [22 × 44.03 (C2H4O repeat unit) + 114.10 (C5H6O3 end groups) + 22.99 (Na+)]. The acetate group most likely came from the vinyl acetate, an impurity found in commercial vinyl acrylate (22). 89 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 8. MALDI-ToF mass spectrum of PEG-diacrylate. (Mn = 1000 g/mol; Mw/Mn = 1.08).

Transesterification of Vinyl Crotonate with PEG Crotonate groups were also successfully introduced at the chain ends of PEG by the transesterification of vinyl crotonate (VC) in the presence of CALB in bulk (Scheme 4).



Scheme 4. Transesterification of vinyl crotonate with PEG (Mn = 1000 g/mol; Mw/Mn = 1.08) in the presence of CALB. [VC] = 8.28 mol/L, [PEG] = 0.89 mol/L; [CALB] = 5.5× 10-4 mol/L. NMR spectroscopy and MALDI-ToF mass spectrometry confirmed the single transesterification product without any side products. Figure 9 shows the 1H and 13C NMR spectra of the transesterification product. The triplet peak of the methylene protons adjacent to the hydroxyl groups shifted downfield from δ=3.50 to δ=4.16 ppm (b) after the reaction. New resonances attributed to the methyl [δ=1.85 ppm (g)] and vinyl [δ=6.90 ppm (e) and δ=5.90 ppm (f)] protons of the crotonate group were observed at the expected positions. The 13C NMR spectrum showed that the carbons connected to the hydroxyl groups in the starting material shifted downfield to δ=63.12 ppm (B) and the carbon signals associated with the crotonate group [δ=166.00 ppm (H), δ=145.26 ppm (F), δ=122.10 ppm (E) and δ=17.55 ppm (G)] appeared at the expected positions, confirming the successful functionalization.

Figure 9. NMR spectra of PEG-dicrotonate (Mn = 1000 g/mol; Mw/Mn = 1.08): (top) 1H NMR spectrum and (bottom) 13C NMR spectrum (solvent: DMSO-d6). The MALDI-ToF mass spectrum includes a peak at m/z 1101.67 which corresponds to the sodium complex of the 22-mer of PEG dicrotonate (Figure 10). The calculated monoisotopic mass for this ion is 1145.73 Da [22×44.03 (C2H4O repeat unit) + 154.08 (C8H10O3 end groups) + 22.99 (Na+)]. In the 91 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


expanded spectrum, a minor series of peaks were distributed 16 m/z units above the main (sodiated) product series; the masses of these products agreed with those of potassiated PEG dicrotonate (1161.68 Da). The calculated monoisotopic mass for the potassiated 44-mer is 1161.70 Da [22x44.03 (C2H4O repeat unit) + 154.08 (C8H10O3 end groups) + 38.96 (K+)].

Figure 10. MALDI-ToF mass spectrum of PEG dicrotonate (Mn = 1000 g/mol; Mw/Mn = 1.08). 92 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Conclusion PEGs were successfully functionalized under solventless conditions via CALB-catalyzed transesterification of vinyl esters. 1H and 13C NMR spectroscopy and MALDI-ToF verified the structure and purity of the products. This method provides a convenient and greener process to functionalize PEGs.


Acknowledgments This work is supported by National Science Foundation NSF under DMR0804878 and the Ohio Board of Regents. We wish to thank The Ohio Board of Regents and NSF for funds used to purchase the NMR (CHE-0341701 and DMR0414599) and MS (CHE-1012636 and DMR-0821313) instruments used in this work.

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