Enzymatic synthesis and characterization of muconic acid‐based unsaturated polymer systems

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University of Groningen

Enzymatic synthesis and characterization of muconic acid‐based unsaturated polymer

systems

Maniar, Dina; Fodor, Csaba; Adi, Indra Karno; Woortman, Albert; Dijken, van, Jur; Loos, Katja

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Polymer International

DOI:

10.1002/pi.6143

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2021

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Maniar, D., Fodor, C., Adi, I. K., Woortman, A., Dijken, van, J., & Loos, K. (2021). Enzymatic synthesis and

characterization of muconic acid‐based unsaturated polymer systems. Polymer International, 70(5),

555-563. https://doi.org/10.1002/pi.6143

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Received: 9 June 2020 Revised: 2 September 2020 Accepted article published: 19 October 2020 Published online in Wiley Online Library: 22 November 2020

(wileyonlinelibrary.com) DOI 10.1002/pi.6143

Enzymatic synthesis and characterization of

muconic acid-based unsaturated polymer

systems

Dina Maniar,

a

Csaba Fodor,

a

*

Indra Karno Adi,

a,b,c

Albert JJ Woortman,

a

Jur van Dijken

a

and Katja Loos

a

*

Abstract

The design of unsaturated aliphatic (co)polyester systems, based on different diester-modiﬁed muconic acid isomers, was per-formed via an eco-friendly pathway by utilizing enzymatic polymerization using Candida antarctica lipase B (CALB) as catalyst. The obtained fully unsaturated oligoesters and polyesters reached lower molecular weights from 2210 to 2900 g mol−1for the cis,cis-(Z,Z)-muconate isomer, and higher molecular weights of up to 21 200 g mol−1for the polymers with cis,trans-(Z,E) iso-meric structures. The obtained (co)polyesters were thoroughly characterized and compared with their saturated polyester ana-logues. The applied biobased catalyst Novozym®435 (an immobilized form of CALB) showed higher selectivity towards the open cis,trans-muconate compared to the more closed-structure cis,cis-muconate. Results of1H NMR analysis showed that alkene functionality is present, and no stereo conformational changes were detected in the resulting polymers. The thermal properties of the muconate-based polyesters showed a glass transition between−7 and 12 °C, and a one-step degradation pro-cess with a maximum rate of weight loss between 415 and 431°C, depending both on the conformation of the applied diester derivatives and on the segment lengths of the polyoxyalkylenes. Mass spectrometric analysis of the resulting saturated and unsaturated polyesters revealedﬁve different microstructures with different terminal end groups, such as ester/hydroxyl, acid/ester, ester/ester and acid/hydroxyl, and cyclic polyesters without functional end groups. Overall, this study demonstrates that enzymatic polymerization is a robust approach for the synthesis of unsaturated polyesters.

Keywords: unsaturated polyester; muconic acid; enzyme catalysis; renewable resources

INTRODUCTION

Commercial unsaturated polyesters (UPEs) are commonly synthe-sized using maleic anhydride as the olefinic monomer due to its low cost, large market scale and high reactivity with various diols.1 However, maleic anhydride is currently synthesized using fossil-resource-derived substrates.2It is also known to be less reactive in subsequent free radical reactions compared to other olefinic monomers such as fumaric acid and muconic acid.3,4The utiliza-tion of carbon-neutral feedstocks for chemical synthesis is one of the most crucial issues in modern green chemistry.5–9In the last few decades great attention has been dedicated to the design of sustainable UPE systems using promising biobased dicarboxylic acid platform chemicals, such as itaconic, fumaric or muconic acids, with a range of various diols/polyols.10–14The double bonds in the polymer backbones offer these materials from renewable feedstock with enhanced functionality in terms of adjustable chemical and mechanical properties, such as glass transition tem-perature, biodegradability, hardness, polarity and strength. These UPEs, for example, are commonly used for composite applications or as thermosetting resins in diverse industrialfields.12, 15–18In

recent work, Beckham and co-workers reported that renewable-based UPEs can have properties that outperform those of fossil-based UPEs in their applications.19They incorporated different muconate derivatives into polyester backbones and found that trans,trans-muconate has the best performance compared to cis,cis-muconate, fumaric acid and maleic anhydride. They found that, as regards storage moduli, one trans,trans-muconate in a

* Correspondence to: C Fodor or K Loos, Macromolecular Chemistry and New

Polymeric Materials, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: cs. fodor80@gmail.com (Fodor); k.u.loos@rug.nl (Loos)

a Macromolecular Chemistry and New Polymeric Materials, Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands b Analytical Chemistry Research Division, Department of Chemistry, Faculty of

Mathematics and Natural Sciences, Bandung Institute of Technology, Bandung, Indonesia

c Current address: Dexa Development Centre, Kawasan Industri Jababeka II, Bekasi, Indonesia

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and

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UPE backbone is equal to approximately three maleic units, and four maleic units in terms of loss moduli.

For the production of unsaturated polymer systems, melt poly-merization by applying conventional inorganic catalysts, such as titanium or tin alkoxides, is currently used.20 However, these methods can lead to undesired side-reactions during polymer for-mation, due to the unsaturated bonds of the reacting compounds, including isomerization, saturation or radical crosslinking. To cir-cumvent these issues and to overcome the difﬁculties of conven-tional chemically catalysed polymerization processes, an attractive alternative pathway, enzymatic polytransesteriﬁcation, has proven to be effective and more eco-friendly for the design of sustainable (un)saturated polymers.21, 22 Additional advantages of enzyme-catalysed polymerizations are the applied mild reaction conditions in terms of temperature, pressure and pH, the enantio- and regio-selectivity as well as the recyclability of the catalysts.21,23,24In addi-tion, they have successfully been applied for the synthesis of vari-ous polymers, such as biobased furan polyester, furan polyamide, aromatic–aliphatic oligo-/polyester, glycopolymer, aliphatic poly-amide and aliphatic–aromatic polyamide.25–37

cis,cis-Muconic acid, also known as 2,4-hexadienedioic acid, is one of the prevalent biobased dicarboxylic acids that are derived from both sugars and lignin.38–40The production of muconic acid in the yeast Saccharomyces cerevisiae and other biotechnological routes has been reported.41,42Moreover, this dicarboxylic acid can be easily converted to its saturated counterparts, to adipic acid by hydrogenation or even to terephthalic acid by isomeriza-tion, followed by a reaction with ethylene and a dehydration process.39,43–45The resulting materials are used for commercial polymer production, such as poly(ethylene terephthalate) or nylon-6,6. The use of muconic acid as a precursor to directly replace monomers is being thoroughly explored. However, the study of its alkene functionality for use as functional monomer replacement has received less attention. The synthesis of unsatu-rated polymers based on muconic acid derivatives via conven-tional metal-catalysed polycondensation or reversible addition– fragmentation chain transfer polymerization has been reported previously.12,46,47 _{To the best of our knowledge, the} enzyme-catalysed copolymerization of muconic acid isomers has not yet been studied.

In the work reported here, aliphatic unsaturated oligoesters and UPEs were designed from various aliphatic diols and isomers of biobased dicarboxylic acid derivatives from muconic acid via enzymatic synthesis. The effects on the molecular weight of the resulting polymers depending on the applied isomers, as well as the thermal properties and microstructure were investigated and compared with the fully saturated aliphatic polyester ana-logues. Utilizing enzymes as catalysts for the synthesis of these unsaturated polymers opens the pathway for their use as func-tional alternatives to petrol-derived materials with improved sus-tainability metrics of the materials as well as the production route.

MATERIALS AND METHODS

Materials

cis,cis-Muconic acid (≥97.0%), 1,4-butanediol (99%), 1,6-hexanediol (99%), 1,8-octanediol (98%), 1,10-decanediol (99%), 1,12-dodecanediol (99%), dimethyl adipate (ADIP;≥99%), dithranol (≥90%), petroleum ether (PE; b.p. 30–40 °C, low-boil-ing-point hydrogen-treated naphtha, puriss.), molecular sieves (type 4 Å), sodium sulfate (Na2SO4;≥99.0%, anhydrous), sodium

carbonate (Na2CO3; ≥99.5%, anhydrous), potassium

trifluoroacetate (KTFA; 98%) and deuterated chloroform (CDCl3; 99.8 at% D) were purchased from Sigma-Aldrich and used without purification. Methanol (MeOH; AR grade) and ethyl acetate (EtOAc; AR grade) were purchased from Biosolve. Sulfuric acid (cc.H2SO4; 98%) was received from BOOM BV. Silica gel (SiliaFlash P60, 40–60 μm) was obtained from Silicycle. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP; >99%) was purchased from TCI Europe and was used without further purification. Novo-zyme® 435 (N435, Candida antarctica lipase B (CALB) immobilized on acrylic resin,>5000 U g−1) was dried under vacuum at 25°C for 24 h prior to use. Diphenyl ether (DPE; 99%) was purchased from Sigma-Aldrich, distilled at 140°C under reduced pressure and stored with 4 Å molecular sieves under inert atmosphere prior to use.

NMR spectroscopy

NMR spectra were acquired with samples in deuterated solvents using a Varian VXR 400 MHz (1H: 400 MHz;13C: 100 MHz) spec-trometer at room temperature. Chemical shifts (⊐) are reported in ppm, whereas the chemical shifts were calibrated to the main solvent residual peaks. The chemical composition and the purity of the compounds were determined using CDCl3as solvent. The collected spectra were analysed using MestReNova (v9.1) (Mestrelab Research SL).

Size exclusion chromatography

The number- and weight-average molecular weights (Mnand Mw) as well as the dispersities (Đ) of the samples were measured rela-tive to narrow-dispersity polystyrene standards (Agilent and Poly-mer Laboratories) in the range 645 to 3.0× 106g mol−1using a SEC system equipped with a Viscotek GPCmax, GPC column oven (VE2585) and two PLgel MIXED-C (5μm × 300 mm) analytical col-umns from Agilent Technologies with a separation range from 200 to 2 × 106 g mol−1 thermostatically controlled to 35°C in CHCl3at aflow rate of 1.0 mL min−1using a Schambeck RI2012 refractive index detector. For sample preparation, purified dry samples (10 mg) were dissolved in CHCl3(3 mL) and after they were completely dissolved they were filtered through a PTFE syringe filter (Minisart SRP 15, Sartorius stedim biotech, PTFE-membranefilter; pore size: 0.45 μm, filter diameter: 15 mm) and analysed using SEC. The collected spectra were analysed using OmniSEC (v5.0) (Malvern).

Matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-ToF MS)

Mass analysis and detection of the produced polymer microstruc-tures were carried out using a Biosystems Voyager-DE PRO spec-trometer in reﬂector/linear and positive mode at an acceleration voltage of 20 kV. Samples were prepared using a premixed mix-ture of dissolved matrix (dithranol, 20 mg mL−1) and cationizing agent (KTFA, 5 mg mL−1) dissolved in HFIP with a volume ratio of 5:1:5. The mixture (0.2–0.4 μL) was subsequently hand-spotted on a stainless-steel target and left to dry in open air. Molecular

weights were calculated from Mn=∑

i NiMi=∑ i Ni and Mw=∑ i NiM2i=∑ i

NiMi, where Mi is the molecular weight of the

chain and Niis the number of chains of that molecular weight. The mass of corresponding oligoesters and polyesters was calcu-lated from Mp= MEG+ (nMRU) + MCI, where MPis the mass of the oligomer or polymer, MEGis the mass of the end groups, n is the number of repeat units, MRUis the mass of the repeat unit and

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Polym Int 2021; 70: 555–563

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MCIis the mass of the counter cation. The MS data were analysed using Data Explorer (v4.9) software (Applied Biosystems). Differential scanning calorimetry

Glass transitions and melting points were measured with a TA Instruments Q1000 differential scanning calorimeter under dry nitrogen atmosphere. The samples were scanned in a tempera-ture range from−10 to 250 °C by heating–cooling–heating cycles using a heating–cooling rate of 10 °C min−1, with isothermal sec-tions between the cycles from 10 min, followed immediately by temperature-modulated DSC measurements in the same temper-ature range at 2 °C min−1 with a temperature modulation of ±0.50 °C for every 60 s. The glass transition temperatures (Tg) taken at the infection points of the speciﬁc heat increase, the melting temperature (Tm) taken as the minimum of the endother-mic peak, the cold crystallization temperature (Tcc) taken as the maximum of the exothermic peak and melting enthalpy (ΔHm) were also determined from the DSC thermograms. The resulting thermograms were evaluated with the use of Universal Analysis 2000 (v4.3A) software (TA Instruments).

Thermogravimetric analysis

Thermal stability and decomposition behaviour measurements were carried out with a TA Instruments D2500. Programmed heat-ing from 35 to 750°C was used for TGA at a heating rate of 10 ° C min−1 under inert atmosphere. The decomposition tempera-ture (Td(max)) of the samples was assigned to the temperatempera-ture of the maximum rate of weight loss. The TGA curves were analysed using TRIOS (v4.1) software (TA Instruments).

Synthetic procedures

Synthesis and separation of isomeric dimethyl muconates

The synthesis was carried out according to the method reported by Frost et al. The cis,cis-muconic acid (5.0 g, 35.2 mmol) was sus-pended in MeOH (150 mL) with catalytic amounts of cc.H2SO4 (0.3 mL) and the mixture was refluxed for 24 h.48 The mixture was cooled, concentrated under vacuum and dissolved in EtOAc. The organic phase was extracted with saturated Na2CO3aqueous solution, followed by washing with brine. The separated organic phase was dried over Na2SO4,filtered and concentrated (5.4 g, 31.8 mmol, 90% yield). A crude racemic mixture of dimethyl muconates (cis,cis and cis,trans) was further purified and sepa-rated by preparatory column chromatography using PE–EtOAc (9/1) eluent mixture. The chemical structures of the separated dimethyl muconate isomers (cis,cis and cis,trans) were confirmed using1H NMR analysis.

cis,cis-Dimethyl muconate (ccMUC): TLC (PE–EtOAc, 9/1): Rf = 0.68; 1H NMR (400 MHz, chloroform-d): ⊐ 7.89 (2H, dd, J= 10.50, 6.09 Hz), 5.99 (2H, dd, J = 10.27, 6.09 Hz), 3.75 (6H, s) ppm;13C NMR (101 MHz, chloroform-d):⊐ 51.68 (C1, C12), 124.05 (C4, C8), 138.13 (C6, C7), 166.21 (C3, C9) ppm.

cis,trans-Dimethyl muconate (ctMUC): TLC (PE–EtOAc, 9/1): Rf = 0.45; 1 H NMR (400 MHz, chloroform-d): ⊐ 8.39 (1H, ddd, J= 15.56, 11.63, 1.02 Hz), 6.64 (1H, t, J = 11.49 Hz), 6.11 (1H, d, J= 15.60 Hz), 5.97 (1H, d, J = 11.34 Hz) ppm;13C NMR (101 MHz, chloroform-d):⊐ 51.82 (C1, C12), 124.25 (C4), 128.66 (C8), 138.62 (C6), 140.62 (C7), 166.54 (C3, C9) ppm.

CALB-catalysed polycondensation by temperature-varied two-stage method

The synthesis was carried out according to our method published previously.31Brieﬂy, in a typical enzyme-catalysed polymerization

experiment pre-dried N435 (15 wt% of the total monomer) and 4 Å molecular sieves (150 wt% of the total monomer) were placed in a round-bottom flask equipped with a magnetic stirring bar under inert atmosphere. The monomers (diesters and diols in defined ratios) and DPE solvent (500 wt% of the total monomer) were added to the catalyst under inert atmosphere. The reaction mixture was allowed to stir slowly (200 rpm) and heated to 85° C for 2 h under nitrogen atmosphere and continuous low stirring speed, followed by reduction of the pressure stepwise to 2 mmHg for the next 22 h. In the next 24 h the temperature was increased to 95°C, followed by an increase to 110 °C for the last 24 h reac-tion time. After the polymerizareac-tion, the reacreac-tion was allowed to cool, and the product was dissolved in CHCl3. N435 and molecular sieves werefiltered out and the mixture was concentrated. The dissolved crude product was precipitated in an excess amount of cold MeOH, and separated by centrifugation, followed by filtra-tion under vacuum on a glassfilter (pore size 3). The resulting polymers were dried and stored under vacuum at room temperature.

Poly(butylmethylene adipate):1H NMR (400 MHz, chloroform-d): ⊐ 4.09 (136H, t, J = 5.07 Hz), 3.67 (4H, t, J = 6.36 Hz), 2.33 (138H, t, J= 6.56 Hz), 1.67 (284H, m), 1.25 (8H, s) ppm.

Poly(hexamethylene adipate):1H NMR (400 MHz, chloroform-d): ⊐ 4.05 (126H, t, J = 6.70 Hz), 3.64 (4H, t, J = 6.50 Hz), 2.32 (125H, t, J= 6.50 Hz), 1.69–1.57 (271H, m), 1.37 (129H, p, J = 3.70 Hz) ppm. Poly(octamethylene adipate):1H NMR (400 MHz, chloroform-d): ⊐ 4.05 (100H, t, J = 6.78 Hz), 3.64 (4H, t, J = 6.60 Hz), 2.32 (99H, t, J= 6.98 Hz), 1.62 (224H, m), 1.31 (211H, m) ppm.

Poly(decamethylene adipate):1H NMR (400 MHz, chloroform-d): ⊐ 4.05 (113H, t, J = 6.80 Hz), 3.63 (4H, t, J = 6.62 Hz), 2.32 (113H, t, J= 6.97 Hz), 1.63 (255H, m), 1.29 (358H, m) ppm.

Poly(dodecamethylene adipate):1H NMR (400 MHz, chloroform-d):⊐ 4.05 (105H, t, J = 6.80 Hz), 3.63 (4H, t, J = 6.60 Hz), 2.32 (106H, t, J= 6.93 Hz), 1.63 (234H, m), 1.29 (446H, m) ppm. Poly(butylmethylene cis,cis-muconate): 1H NMR (400 MHz, chloroform-d):⊐ 7.89 (3H, t, J = 10.16 Hz), 5.98 (3H, t, J = 7.18 Hz), 4.19 (5H, m), 3.76 (3H, s), 3.65 (4H, m), 4.19 (63H, s), 1.27 (8H, d, J= 11.38 Hz), 0.85 (18H, dd, J = 11.34, 9.03 Hz) ppm. Poly(hexamethylene cis,cis-muconate): 1H NMR (400 MHz, chloroform-d):⊐ 7.88 (30H, t, J = 8.87 Hz), 5.97 (30H, t, J = 8.21 Hz), 4.14 (50H, t, J= 6.64), 3.75 (4H, s), 3.64 (4H, t, J = 6.62 Hz), 1.69 (44H, m), 1.42 (56H, s) ppm. Poly(octamethylene cis,cis-muconate): 1H NMR (400 MHz, chloroform-d):⊐ 7.88 (24H, dd, J = 8.06, 2.22 Hz), 5.97 (28H, dd, J= 8.17, 1.97 Hz), 4.14 (34H, t, J = 6.69), 3.75 (6H, s), 3.65 (4H, t, J= 6.62 Hz), 1.69 (32H, q, J = 6.70 Hz), 1.54 (36H, s), 1.42 (78H, d, J= 11.52 Hz) ppm. Poly(decamethylene cis,cis-muconate): 1H NMR (400 MHz, chloroform-d):⊐ 7.88 (28H, dd, J = 8.21, 1.87 Hz), 5.97 (24H, dd, J= 8.05, 2.24 Hz), 4.14 (34H, t, J = 6.69), 3.75 (6H, s), 3.65 (4H, t, J= 6.62 Hz), 1.66 (30H, p, J = 7.07, 6.65 Hz), 1.33 (106H, m) ppm. Poly(dodecamethylene cis,cis-muconate): 1H NMR (400 MHz, chloroform-d):⊐ 7.88 (28H, dd, J = 8.33, 1.53 Hz), 5.97 (24H, dd, J= 8.28, 1.65 Hz), 4.14 (26H, t, J = 6.63), 3.75 (4H, d, J = 1.40 Hz), 3.64 (4H, m), 1.66 (52H, q, J= 7.01 Hz), 1.27 (252H, m) ppm. Poly(hexamethylene cis,trans-muconate): 1H NMR (400 MHz, chloroform-d):⊐ 8.39 (12H, dd, J = 15.64, 11.61 Hz), 6.63 (14H, t, J = 11.46 Hz), 6.10 (14H, d, J = 15.57 Hz), 5.96 (14H, d, J= 11.38 Hz), 4.17 (60H, qd, J = 6.86, 5.41, 2.41 Hz), 3.64 (4H, t, J= 6.53 Hz), 1.69 (63H, m), 1.42 (97H, m) ppm. Poly(octamethylene cis,trans-muconate): 1H NMR (400 MHz, chloroform-d): ⊐ 8.39 (8H, dd, J = 15.09, 11.98 Hz), 6.63 (9H, t,

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J= 11.51 Hz), 6.10 (9H, d, J = 15.57 Hz), 5.96 (10H, d, J = 11.38 Hz), 4.17 (38H, t, J= 6.78 Hz), 3.64 (4H, t, J = 6.64 Hz), 1.67 (15H, m), 1.33 (197H, m) ppm. Poly(decamethylene cis,trans-muconate): 1H NMR (400 MHz, chloroform-d): ⊐ 8.39 (8H, dd, J = 15.09, 11.98 Hz), 6.63 (9H, t, J= 11.51 Hz), 6.10 (9H, d, J = 15.57 Hz), 5.96 (9H, d, J = 11.38 Hz), 4.16 (40H, t, J= 6.78 Hz), 3.64 (4H, t, J = 6.64 Hz), 1.67 (42H, m), 1.33 (136H, m) ppm. Poly(dodecamethylene cis,trans-muconate):1H NMR (400 MHz, chloroform-d):⊐ 8.38 (20H, dd, J = 15.59, 11.67 Hz), 6.63 (23H, t, J = 11.53 Hz), 6.10 (22H, d, J = 15.56 Hz), 5.96 (24H, d, J= 11.37 Hz), 4.16 (99H, t, J = 6.80 Hz), 3.64 (4H, t, J = 6.64 Hz), 1.68 (103H, m), 1.27 (426H, m) ppm.

Control polycondensation reactions

Polymerization reactions similar to the applied temperature-varied two-stage method were carried out without CALB.

RESULTS AND DISCUSSION

Monomer synthesis and separation

Linear polymeric materials with unsaturated bonds in their back-bones are desirable materials due to their possible further modi ﬁ-cations, functionalization or even crosslinking. First, monomers were produced and isolated via conversion of cis,cis-muconic acid by esteriﬁcation reactions to racemic muconic acid diester com-pounds followed by cis,cis and cis,trans isomeric arrangement sep-aration using column chromatography (Scheme 1). Only a small amount (<3%) of mucolacton was isolated (TLC (PE–EtOAc 9/1):

Rf= 0.43). NMR spectra of the racemic cis,cis and cis,trans isomers of muconates and mucolacton are shown in Fig. S1 (supporting information).

Enzymatic polymerization

The enzymatic polycondensation reactions were conducted at an optimal temperature of 85°C and under inert atmosphere, fol-lowed by dynamic stepwise-reduced pressure.31 In the case of low-carbon-number diols with low boiling points, this procedure caused elimination/loss of diols in the later stages, producing only low amounts (yield below 2%) and low-molecular-weight com-pounds. In order to fully understand the enzymatic polymeriza-tion of the muconate isomers (cis,cis- and cis,trans-muconate), the saturated derivatives of these oligoester/polyester copolymer systems were also synthesized and investigated utilizing dimethyl adipate as comonomer and applying the same enzymatic conditions.

Table 1 summarizes the synthetic conditions as well as the molecular weights of the polymers obtained by the enzymatic condensation polymerizations of the different aliphatic diesters and diols with various methylene numbers between 4 and 12 (Scheme 2). The chemical structures of the produced saturated and unsaturated polyesters were conﬁrmed using NMR analysis (Figs S2–S4, supporting information). The1H NMR results show that the alkene functionality is still intact and no stereo conforma-tional changes were detected in the resulting polymers. This shows that undesired side-reactions such as isomerization, satura-tion or radical crosslinking during the polymerizasatura-tion were pre-vented by utilizing the enzymatic approach.

Scheme 1. Esteriﬁcation reaction implemented for production of racemic muconates (cis,cis and cis,trans isomers of muconates) and mucolacton after

isomerization of muconates.

Table 1. Synthetic conditions for polymerization of saturated and unsaturated aliphatic diesters and diols with various carbons between 4 and 12

and molecular weight of polymers obtained

Entry Diester Diol (C number) Diester/diol ratio Yield

NMR GPC

Mn(g mol−1) Mn(g mol−1) Mw(g mol−1) Ð

1 ADIP 4 50/50 55 6870 5500 9020 1.64 2 ADIP 6 50/50 66 1400 16 100 34 800 2.16 3 ADIP 8 50/50 83 13 500 15 800 31 600 2.00 4 ADIP 10 50/50 91 9100 15 800 32 600 2.06 5 ADIP 12 50/50 94 8700 16 200 35 200 2.17 6 ccMUC 4 50/50 <5 680 — — — 7 ccMUC 6 50/50 8 1690 1800 2210 1.23 8 ccMUC 8 50/50 8 1910 1960 2490 1.27 9 ccMUC 10 50/50 10 2420 2300 3290 1.42 10 ccMUC 12 50/50 7 2050 2080 2900 1.39 11 ctMUC 4 50/50 _<2 _— _— _— _— 12 ctMUC 6 50/50 12 2160 3040 3610 1.49 13 ctMUC 8 50/50 13 2920 4760 8000 1.68 14 ctMUC 10 50/50 15 2980 5970 10 820 1.81 15 ctMUC 12 50/50 17 5500 6780 21 200 3.16

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The saturated diester-containing samples (Table 1, entries 1–5) produced polymers with Mnfrom 5500 to 16 200 g mol−1with Ð of 1.64 to 2.17, while the unsaturated diesters, due to the steric hindrance of the monomers, could only form oligomers or low-molecular-weight polymers, with molecular weights up to 2300 and 6780 g mol−1, depending on the isomeric structure (Table 1, entries 6–15). The steric hindrance is caused by the dou-ble bonds in the muconate isomers. Together with the diols, the ﬂexibility of the formed macromolecules is decreased, and may not be able toﬁt in the active pocket of the CALB enzyme. There-fore, the propagation of the resulting oligomers cannot proceed any further. Interestingly, by comparing the different isomers of the muconates, it can be seen that the cis,trans isomers are able to form higher molecular weight polymers. This can be explained by the more open structure of the cis,trans isomers, which results in better accessibility of the functional groups to the CALB active site.31In contrast, the closed structure of the cis,cis isomers is lim-iting to the propagation.

The general trend in molecular weights in relation to the diols used is in agreement with the fact that CALB enzymes exhibit higher afﬁnity towards longer methylene chain-bearing mono-mers; thus increasing molecular weights are expected for diols with increasing methylene numbers.49 _{The lower yields of the} UPE compounds are mainly due to the lower solubility in the applied solvent during the puriﬁcation process compared to the saturated counterparts.

The microstructures and end groups of the resulting oligoesters and polyesters were analysed using MALDI-ToF MS. By evaluating the set of peaks of the corresponding MALDI-ToF MS spectra, the fully saturated polyester samples (Table 1, entries 1–5) revealed ﬁve different microstructures (Fig. S9, supporting information). Similarly, as presented in Fig. 1, the end group analysis of the resulting UPEs derived from cis,cis-muconate (Table 1, entries 6–10) revealed ﬁve different polyester species, with different ter-minal end groups, such as ester/hydroxyl, acid/ester, ester/ester and acid/hydroxyl, and cyclic polyesters without functional end groups (see also Fig. S10, supporting information). As previously reported, the occurrences of acidic end groups are due to the cat-alytic hydrolysis reaction of the applied lipase enzyme.25,26,35The

MALDI-ToF MS spectra of cis,trans-muconate (Table 1, entries 11–15), on the other hand, revealed only two to three microstruc-tures (see Fig. S11, supporting information). In summary, MALDI-ToF MS analysis of all samples further supports the fact that no additional side products were formed from the enzymatic polymerization.

Investigation of the thermal properties of the compounds showed that the synthesized unsaturated oligoesters and polyes-ters are amorphous materials, due to the retarded segmental motion of the diester moieties, which disrupts the crystalline pack-ing of the polyol segments (Fig. 2). Tgof the muconate-based linear polyesters varied between−7.1 and 12.5 °C for the cis,cis-muconate derivatives, as well as−1.6 and 9.8 °C for the cis,trans-muconate derivatives, slightly depending both on the conformation of the applied diesters and on the polyol segment lengths. These mea-sured transition values of the muconates are in accordance with the results of Beckham and co-workers for various renewable UPE systems after incorporation of 2.2 to 12.8 mol% muconic acid into succinic-based polyesters.12

To study the thermal stability of the polymers, TGA measure-ments were carried out. Figure 3 shows the TGA (DTGA) traces of the cis,cis- and the cis,trans-muconate-based polyesters obtained under nitrogen atmosphere. The TGA traces show slight weight loss (less than 10%) below 200°C, due to the evaporation of the absorbed water from the polyesters. This solvent content of the samples was taken into account for the comparability of the results. The TGA and DTGA traces show that the thermal degrada-tion of the UPEs occurs in a one-step degradadegrada-tion process with a sharp weight loss between 340 and 500°C with a maximum rate of weight loss between 415 and 421°C (cis,cis-muconate-based samples) and from 425 to 431°C (cis,trans-muconate-based sam-ples), depending slightly on the methylene numbers of the diols used. The degradation process leads to near-complete decompo-sition with a low char residue. Based on the TGA results, the inves-tigated unsaturated aliphatic polyesters showed high thermal stabilities.

The competitive incorporation of unsaturated and saturated diesters into polyester systems during an enzymatic polymeriza-tion was investigated to study if one of the monomers is

Scheme 2. Polymerization reaction of (un)saturated (dimethyl adipate and cis,cis and cis,trans muconates) diesters with polyoxyalkylenes (with various methylene numbers between 4 and 12).

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polymerized preferentially. The ratio of the two esters (M1/M2) was varied, keeping the diol ratio the same by using diols with methylene number of 6 and 8 for the different copolymers (Table 2). One can see that the ratios of the different diesters in the obtained polymer are close to the expected values/the mono-mer feed, but in all cases, slight differences in the incorporation

can be observed. By investigating the incorporation of the unsat-urated and satunsat-urated diesters into the polyesters, the isomer with the more open conformation showed a higher afﬁnity for poly-merization during the competitive reactions between the more ﬂexible and the sterically hindered counterparts. The molecular weight of the resulting copolyester systems showed an increasing

(a)

500 2000 3500 5000 6500 0 20 40 60 80 100 n=16 n=14 n=12 n=10 n=8 n=6 n=4 Int ens ity ( % ) m/z

(b)

1300 1400 1500 1600 1700 1800 0 20 40 60 80 100 B A E E A C D D C [ [Mc ] n +Na ] + [ [Mc ] n +Na ] + [[Mc] n+K ] + [ [Mc ] n +K ] + [( HO -[M] n-COO H ) +K ] + [( HO -[M] n-COO H ) +K ] + [( CH 3O -[M] n-CO O H ) +K ] + [( CH 3O-[M] n-C OO H ) +K ] + [( CH 3O-[M ] n -O H ) +K ] + [( CH 3O-[M ] n -O H ) +K ] + [( CH 3O-[M ] n -O C H3 ) +K ] + [( CH 3O-[M ] n -O CH 3 ) +K ] + In ten si ty (% ) m/z 280 Da B

(c)

Figure 1. (a) MALDI-ToF MS spectrum of unsaturated oligoester (Table 1, entry 9) produced via enzymatic polymerization, (b) magniﬁed part with

detailed peak interpretation and (c) microstructures of the obtained oligoester.

Figure 2. DSC thermograms (second heating cycles) of (a) muconate (cis,cis conformation) (Table 1, entries 7–10) and (b) muconate (cis,trans

conforma-tion) based linear polyesters (Table 1, entries 12 to 15).

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trend depending on the incorporation of the saturated counter-parts in the aliphatic polymers. In the case of 1,8-octanediol monomers the molecular weights are increased for the mixed

systems compared to the lower methylene number diol polymers, due to the higher afﬁnity of the enzyme towards the longer chain monomers.49

Figure 3. (a, c) TGA and (b, d) DTGA traces of muconate (cis,cis conformation) (Table 1, entries 7–10) and muconate (cis,trans conformation) based linear

polyesters (Table 1, entries 12–15).

Table 2. Synthetic conditions for polymerization of adipate- and muconate-containing linear diesters with different polyoxyalkylenes with carbon

numbers of 6 and 8 and molecular weight of polymers obtained

Entry Diester Diol (C number) Feed Copolymer Yield GPC

M1 M2 Diester (M1/M2)/diol ratio Diester (M1/M2)/diol ratio Mn(Da) Mw(Da) Ð

16 ADIP ccMUC 6 (12.5/37.5)/50 (17.6/31.5)/51.9 10 2610 3350 1.28 17 ADIP ccMUC 6 (25/25)/50 (26.3/23.1)/50.6 29 4350 9030 2.26 18 ADIP ccMUC 6 (37.5/12.5)/50 (34.5/16.3)/49.2 64 11 760 14 510 1.23 19 ADIP ccMUC 8 (12.5/37.5)/50 (21.1/28.9)/50.1 43 520 10 200 1.96 20 ADIP ccMUC 8 (25/25)/50 (25.2/23.0)/51.8 37 8550 21 080 2.36 21 ADIP ccMUC 8 (37.5/12.5)/50 (37.3/12.8)/49.9 29 5850 11 650 2.04 22 ADIP ctMUC 6 (12.5/37.5)/50 (16.7/32.2)/51.1 21 8450 19 400 2.3 23 ADIP ctMUC 6 (25/25)/50 (29.1/21.9)/49.0 34 9320 18 690 2.03 24 ADIP ctMUC 6 (37.5/12.5)/50 (37.9/11.3)/50.8 54 15 180 31 480 2.79 25 ADIP ctMUC 8 (12.5/37.5)/50 (15.4/34.3)/50.3 34 9260 16 210 1.75 26 ADIP ctMUC 8 (25/25)/50 (28.0/21.9)/50.0 50 9910 48 070 4.85 27 ADIP ctMUC 8 (37.5/12.5)/50 (41.3/10.1)/48.6 32 15 180 31 480 2.79

561

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The DSC thermograms of the fully saturated semi-crystalline polyester of adipate and 1,6-hexanediol (Fig. S12, supporting information) revealed a sharp melting peak around 58°C, with a melting enthalpy (ΔHm) of 85 J g−1. With the incorporation of muconates into the saturated aliphatic polyester systems, the crystallinity of the resulting polyesters was suppressed. The aliphatic polyesters with lower amounts of sterically hindered moieties showed decreased crystallinities compared to the fully saturated aliphatic polyester samples (Fig. S13, supporting infor-mation) as a result of disruption of the packing of the crystallites formed in the samples. This effect is more pronounced with incorporation of an increased amount of retarded-motion moie-ties (Fig. 4). The Tg values of these samples showed also an increasing tendency towards broadening the transition, due to the different segment motions of the randomly incorporated moieties in the investigated materials. A similar effect was also observed in the case of poly[(butylene succinate)-co-muconate]

copolymers with 2.2 to 12.8 mol% muconic acid contents in the polyesters.12

CONCLUSIONS

The focus of the work was directed towards the possibility of designing unsaturated aliphatic oligoesters and polyesters via enzyme-mediated step-growth polymerization. Different unsatu-rated oligoesters and polyesters were successfully synthesized in a CALB-catalysed polycondensation using a temperature-varied two-stage method from polyoxyalkylenes with various methylene numbers and two different muconate isomer structures. Polymer-ization of the fully saturated monomer counterparts resulted in polymers with molecular weights up to 35 200 g mol−1, while the unsaturated diesters, due to the steric hindrance of the mono-mers, could only form lower molecular weight products. Polyes-ters with lower molecular weights up to 2900 g mol−1 and

Figure 4. DSC thermograms (second heating cycles) and theirﬁrst derivatives of saturated/unsaturated polyester samples with various ratios of diesters:

(a) adipate/muconate (cis,cis conformation) with 1,6-hexanediols (Table 2, entries 16–18); (b) adipate/muconate (cis,cis conformation) with 1,8-octanediols

(Table 2, entries 19–21); (c) adipate/muconate (cis,trans conformation) with 1,6-hexanediols (Table 2, entries 22–24); (d) adipate/muconate (cis,trans

con-formation) with 1,8-octanediols (Table 2, entries 25–27).

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higher molecular weights up to 21 200 g mol−1were obtained for the cis,cis-muconate and cis,trans-muconate isomer structures, respectively. This can be explained by the higher selectivity of CALB towards the open structure of cis,trans-muconate compared to the more closed structure of cis,cis-muconate.1H NMR analysis of the resulting polymers showed that the unsaturated bond functionality was still intact and no stereo conformational changes were detected after the polymerization. By utilizing an enzymatic approach, undesired side-reactions such as isomeriza-tion, saturation or radical crosslinking can be prevented. In addi-tion, the MALDI-ToF MS analysis of all samples further suggested that no additional side products were formed from the enzymatic polymerization. All obtained muconate-based polyesters exhibit a glass transition between−7.1 and 12.5 °C, and a one-step degra-dation process with a maximum rate of weight loss between 415 and 431°C, depending both on the conformation of the applied diester derivatives and on the segment lengths of the polyoxyalkylenes. Muconic acid and its different isomeric struc-tures are promising dicarboxylic acid renewable platform mole-cules and the produced materials will pave the way to potential applications for UPE resins or photosensitive coatings.

ACKNOWLEDGEMENTS

The authors are grateful for theﬁnancial support of the Indone-sian Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan LPDP) and the European Research Area Industrial Bio-technology (ERA IB) from the European Commission's Sixth Framework Programme (FP6) Oxypol project.

CONFLICT OF INTEREST

The authors declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses or inter-pretation of data, in the writing of manuscript or in decision to publish the results.

SUPPORTING INFORMATION

Supporting information may be found in the online version of this article.

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