Synthesis and Enzymatic Degradation of Soft Aliphatic Polyesters.

Full Paper

Synthesis and Enzymatic Degradation of Soft Aliphatic Polyestersa Viola Buchholz, Seema Agarwal, Andreas Greiner* Novel aliphatic enzymatically degradable polyesters with short alkyl side chains for tuning crystallinity are presented in this work. The intrinsic problem of aliphatic polyesters is their brittleness and tendency to crystallize. This was modulated by the synthesis of random copolyesters based on aliphatic linear monomers, adipic acid, 1,5-pentanediol and monomers with aliphatic branches, such as 2-butyl-2-ethyl-1,3-propanediol by polycondensation. The resulting copolyesters were crystalline, wax-like or had liquid texture with varied mechanical properties and enzymatic degradability depending upon the copolymer composition. Such polyesters are of significant interest for a wide range of possible applications such as controlled drug delivery, agricultural applications and as packing materials.

1. Introduction Many applications in biomedicine and agriculture require biodegradable polymers. In most cases, aliphatic polyesters, such as poly(lactid acid) (PLA),[1,2] poly(ecaprolactone) (PCL),[3,4] or poly-3-hydroxybutyrate (P3HB),[5,6] are used. These polymers are semicrystalline and highly brittle. Nevertheless, there are many applications that require mechanically soft biodegradable polymers. One approach is to synthesize soft materials with low glass transition temperatures by reduction of the crystallinity of a linear polymer. This can be achieved by the introduction of side chains to the polymer backbone.

A. Greiner, V. Buchholz, S. Agarwal Macromolecular Chemistry II and Bayreuth Center for Colloids and Interfaces, University of Bayreuth, D-95440 Bayreuth, Germany a Supporting Information is available from the Wiley Online Library or from the author.

Macromol. Biosci. 2016, 16, 207–213 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The variation of the properties of PCL was reported in the past by the introduction of side chains to the polymer. Therefore, e-decalactone was polymerized by ring-opening polymerization. The appearance in form of a viscous liquid and the absence of a melting point indicated the amorphous properties of poly-decalactone with the remaining butyl side chains in comparison to the semicrystalline PCL.[7,8] A higher control of the polymer properties was achieved by varying the amount of side chains. This concept was proved on polyesters synthesized by enzymatic copolymerization of biobased monomers, such as 12-hydroxydodecanoic acid and methyl-12-hydroxystearate. The thermal and mechanical properties of the resulting aliphatic polyester with hexyl side chains can be varied by the copolymer ratio between solid and liquid materials.[9] The influence of the side chain length was also analyzed.[10] Polyesters with different side chain lengths between C6 and C16 based on fatty acids were synthesized and it was revealed that the thermal properties are highly dependent on the side chain length. The longer the chains are, the lower the melting points.

wileyonlinelibrary.com

DOI: 10.1002/mabi.201500279

207

V. Buchholz, S. Agarwal, A. Greiner www.mbs-journal.de

Here, we demonstrate the transfer of the concept of introducing side chains into a linear polymer to adjust thermal and mechanical properties of polyadipates, which can be synthesized via polycondensation. Particular focus is tailoring of their biodegradability in combination with low glass transition temperatures, which will open the pathway to novel applications. Starting with poly(pentamethylene adipate) as a linear polymer synthesized from adipic acid and 1,5-pentanediol, this was modified by copolymerization with 2,2-butyl-ethyl-1,3-propanediol to include ethyl and butyl side chains. The enzymatic degradability of the new copolyesters was tuned by the copolyester composition which offers a wide degradation scale for applications such as soft material for agricultural and biomedical uses.

2. Experimental Section 2.1. Materials Adipic acid (Fluka 99%) was recrystallized from water. 1,5-pentanediol (Fluka 97%), tetrahydrofuran (THF) and isohexane were distilled before use. 2,2-Butyl-ethyl-1,3-propane€ ls), esterase diol (Aldrich Sigma 99%), titanium(IV) butoxide (Hu EL-01 (suspension, enzyme amount 20.4 mg ml1, origin: thermomyces lanuginosus, ASA-Spezialenzyme GmbH), tris(hydroxymethyl)-aminomethane (TRIS; Roth 99.3%), and poly(e-caprolactone) (PCL CapaTM 6800, molecular weight 80 000, Perstorp) were used as received.

2.2. Analytical Methods 1 H-NMR and 13C-NMR spectra were recorded on a Bruker Ultrashield 300 operating at a frequency of 300 MHz for 1H and 75 MHz for 13C, respectively. Deuterated-chloroform was used as a solvent. MestReNova (MestreLab Research S.L., version 6.1.0-6224) was used to evaluate the spectra. The remaining solvent signal at 7.26 ppm (1H NMR) and 77.0 ppm (13C NMR), respectively, was used as an internal reference. The determination of the molar mass was carried out by gel permeation chromatography (GPC) with chloroform as the eluent at a flow rate of 0.5 mL min1 at 20 8C, a precolumn PSS SDV (particle size 5 mm) and a column PSS SDV XL linear (particle size 5 mm) calibrated against polystyrene standards (PSS) using a PSS SECcurity RI detector. The GPC data were analyzed by the software PSS WinGPC Unity, Build 1321. Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere on a Netzsch TG 209 F1 Libra with a heating rate of 20 K min1 from 25 to 800 8C. Differential scanning calorimetric (DSC) characterization was performed under a nitrogen atmosphere on a Mettler Toledo 821c DSC system calibrated with indium and zinc standards. The samples were heated from 100 to 150 8C with a heating rate of 20 K min1 and cooled from 150 to 100 8C with a cooling rate of 20 K min1. Second heating runs were recorded for the analysis of glass transition temperatures (Tg) and melting temperatures (Tm). The center of the transition area was used for Tg and the onset

208

temperature was determined for Tm. The mechanical properties of polyester films prepared by the solvent-cast method were measured with a Zwick/Roell BT1-FR 0.5TN-D14 machine equipped with a 200 N KAF-TC load sensor. The stretching rate used was 50 mm min1 with a small preload of 0.05 N mm1. All specimens were cut into dog bone shape with a length of 30 mm and a width of 2 mm. The measurements were carried out at room temperature (20 8C). The Young’s modulus was calculated from the initial slope of the stress–strain curve and averaged for at least five samples. Polyester films of 10 10 0.2 mm3 were prepared by heat press at 40 8C for further investigations and kept under ambient temperature for at least 2 weeks in order to attain equilibrium crystallinity. The films were placed in small glass containers with 4 mL of buffer solution (pH 9, TRIS HCl, tris(hydroxymethyl)-aminomethane with hydrochloride acid, 1M) for enzymatic degradation studies. The degradation of the polyester films was started by the addition of the enzyme suspension (0.25 mL). The system was shaken slowly at 20 8C. After different time intervals, films were rinsed with distilled water and freeze-dried until constant mass was reached. The remaining mass was determined for each sample by comparison of the dry mass (md) with the initial mass (m0), as shown in Equation (1). The mass change was recorded as the average of the three individual degraded samples. remaining mass in % ¼ md =m0 100%

ð1Þ

Scanning electron microscopy (SEM) images were recorded on a LEO 1530 (Gemini) using the software SmartSEM (Carl Zeiss SMT Ltd, version 5.4.5.0) and a SE2 detector. The accelerating voltage was 2 kV. The samples were coated with 2.0 nm of platinum prior to the measurements. A Cressington sputter coater (208HR, 40 mA, 0.06 mbar) equipped with a Cressington thickness controller (mtm-20) was used. Wide-angle X-ray diffraction (WAXD) was measured using a Bruker AXS D8 Advance equipped with a vertical goniometer using Cu Ka radiation. The data were acquired in the 2u interval of 108–808 in steps of 0.058. The diffractograms obtained were analyzed after a baseline correction to reduce the intensity of the primary beam. The degree of crystallinity was determined as a ratio of the integral of the crystalline reflections and the complete diffraction curve including the amorphous halos.

2.3. General Procedure for Synthesis of Polyesters (4, 5a–h). The diacid 1 and the diols 2 and 3 were mixed under an Argon atmosphere in a flame-dried Schlenk flask equipped with a septum in the quantities given in Table 1. A catalytic amount of titanium(IV) butoxide (30 mL) was added after the mixture had been stirred for 4 h at 180 8C. After closing the apparatus, the temperature was increased to 220 8C and the melt stirred under vacuum (102 mbar) for an additional 40 h. The polyesters obtained were dissolved in THF after cooling to room temperature and precipitated in cold iso-hexane. Yields, molecular masses, molarmass dispersities, Tg and Tm for polyesters 4 and 5a–h are given in Table 1. The NMR and FTIR spectra for the copolymers were almost identical with only minor changes in signal intensities occurring. Typical values are provided for copolymer 5h.


www.MaterialsViews.com

Synthesis and Enzymatic Degradation of Soft Aliphatic Polyesters www.mbs-journal.de

Table 1. Molar ratios of the monomers adipic acid/1,5-pentanediol/2,2-butyl-ethyl-propane-1,3-diole (1/2/3) in feed and in polymer, isolated yields, molar mass results and characteristics from DSC for copolymers 4, 5a–h.

Molar ratio In feed

In polymera)

Yield[%]

M n b)

D̵ M[M w =Mn ]b)

Tgc)[8C]

Tmc) [8C]

4

1/1/0

1/1/0

85

43 000

1.99

55

33

Polyester

5a

1/0.975/0.025

1/0.974/0.026

93

41 800

1.96

55

31

5b

1/0.95/0.05

1/0.95/0.05

90

31 900

1.82

55

26

5c

1/0.925/0.075

1/0.927/0.073

91

59 200

1.77

54

19

5d

1/0.9/0.1

1/0.91/0.09

95

44 400

1.87

54

12

5e

1/0.85/0.15

1/0.86/0.14

90

39 200

1.95

57

8

5f

1/0.8/0.2

1/0.8/0.2

95

53 600

2.17

56

–

5g

1/0.75/0.25

1/0.76/0.24

88

28 500

1.85

55

–

5h

1/0.5/0.5

1/0.5/0.5

81

30 400

2.00

50

–

a) determined via 1H-NMR; b)determined via chloroform GPC and PS standard calibration; c)obtained from second DSC heating trace, center values for Tg, onset values for Tm.

1 H NMR. (300 MHz, CDCl3) d ¼ 4.08 (t, J ¼ 6.6 Hz, 4H), 3.92 (s, 4H), 2.34 (s, 2H), 1.65 (m, 12H), 1.50–1.12 (m, 10H), 0.91 (t, J ¼ 6.9 Hz, 3H), 0.83 (t, J ¼ 7.5 Hz, 3H). 13 C NMR. (75 MHz, CDCl3) d ¼ 173.36, 173.30, 173.18, 173.12, 65.80, 64.15, 39.31, 33.88, 33.84, 30.37, 28.27, 24.70, 24.38, 23.65, 23.39, 22.42, 14.02, 7.17. FTIR. (ATR) n ¼ 3446 w, 2944 b, 2868 w, 1730 vs, 1461 m, 1419 w, 1385 w, 1354 w, 1240 m, 1165 s, 1139 s, 1072 m, 989 w, 917 w, 775 w, 735 w, 602 w.

Scheme 1. Polycondensation of random copolyesters with aliphatic side chains. The compositions are given below (Table 1).

3. Results and Discussion 3.1. Polymer Synthesis Random aliphatic copolyesters with varying amount of aliphatic side chains were synthesized by titanium(IV) butoxide copolycondensation of adipic acid (1), 1,5pentanediol (2) and 2,2-butyl-ethyl-propane-1,3 diol (3), according to Scheme 1. Isolated yields of the copolycondensations were in the range above 80% (Table 1). Deviations from 100% were explained by losses in work-up procedure and minor sublimation during polycondensation. All polyesters synthesized displayed unimodal dispersity around 2. The experimental copolyester compositions were determined by 1H-NMR spectroscopy via integration of the signal corresponding to the methylene groups next to the ester function (Figure 1, 3.89 and 4.05 ppm, marked with 1,5 and 12,14 for the diol compounds and 2.31 ppm with label 7,10 for the diacid, respectively). Except for some minor variations, the experimental composition corresponded well to the feed composition of


the comonomers. Concerning the microstructure, no fine splitting of NMR signals was detected, therefore atactic polymers were assumed.

3.2. Thermal Properties Different amounts of side chain monomer influenced the thermal behavior of the polyesters significantly. All polyesters showed glass transition temperatures in common, around 55 8C in DSC traces (Figure 2a). Concerning the side chain influence, polyesters with a low amount were crystalline (4, 5a–e). Their melting temperature decreased with an increasing amount of the side chain repeat unit (Table 1). Their crystallinity was obviously gradually suppressed by the side chains and was supported by decreasing enthalpy of fusion (Figure 2b). Polyesters with high amount of side chains (5f–h) are amorphous. The results from DSC were provided by the appearance of the


209


Figure 1. 1H and 13C NMR spectra of copolymer 5h (CDCl3, 300 MHz, 75 MHz, respectively).

copolyesters at room temperature; crystalline polyesters were solids (4 and 5a, e), while low crystalline and amorphous polyesters were wax-like or even liquid (5f–h).

Focusing on the whole stress–strain curves, the increasing amount of side chains lead to an increasing maximum elongation, except for the polyester 5e which had a very low maximum strain. This basic tendency to rise was also approximately visible for the ultimate tensile strength.

3.3. Mechanical Properties The mechanical properties and influence of side chains were analyzed for the copolyesters 4 and 5a–e. According to their wax-like and liquid appearance, polyesters with a high amounts of side chains, 5f–h, were not analyzed. The Young’s modulus for polyester 4 is about 0.19 GPa. Furthermore, the modulus decreased continuously with the increasing amount for the polyesters with side chains (Figure 3). The copolymer with 15% side chain, 5e, exhibited a value of only 0.06 GPa. Thus, the remaining values corresponded to modulus values for other aliphatic polyesters, such as poly(e-caprolactone) with 0.15 GPa.

3.4. Enzymatic Degradation The degradation behavior of the polyester films 4 and 5a–e were analyzed by keeping samples of each film in buffered enzymatic solution and the remaining mass was determined after a defined time interval. The chosen pH value of 9 corresponds to the activity range of the used esterase. In summary, a decrease of the remaining mass was detected for all samples up to almost full dissolution for polymers 5a–e (Figure 4). The results prove that the introduction of side chains into the polymer backbone leads to an increased

Figure 2. DSC traces (second heating runs) of polyesters 4, 5a–h (a) and corresponding enthalpy of fusion as a function of side chains determined, obtained by integration of DSC melting peaks for polyesters 4, 5a–e (b).

210




Figure 3. Stress–strain curves of polyesters 4, 5a–e (a) and corresponding Young’s modulus as a function of side chains obtained from initial slope of stress–strain curves (b).

degradation rate compared to the linear polyester. The slight faster degradation of polymer 5b is an effect of the fluctuation of the enzyme activity. The differences to the other synthesized polyesters with side chains are in the range of the measuring accuracy. By contrast, a sample of each polymer film was placed in pure buffer solution and no change in the remaining mass was detected over a time period of 72 h. Therefore, no hydrolytic degradation was assumed. Additionally, PCL was analyzed under identical conditions as a comparison which is known to be degradable in enzymatic solutions.[11] The data obtained show comparable degradation behavior of PCL and the novel synthesized polyesters containing different amounts of side chains. The GPC analysis of the degraded polymer films showed no changes in molar mass distribution caused by enzyme exposure. Therefore, surface erosion is assumed. The SEM

images of the surface of the polymer films supported this assumption. In all cases, the smooth surface of the films before the enzymatic degradation changes into a rough area with holes and slots after 16 h in the buffered enzyme solution. No changes are visible in the case of a pure buffer system (Table 2). The structural class of polyester and the degradation behavior is intensively investigated.[11–18] The enzymatic degradation of other poly(adipates) were analyzed in detail by Gan et al.[12] The degradation behavior depended on the crystallinity, the crystal size and the crystal structure, which influenced the degradation rate and resulting morphological changes. The occurrence of sheaf-like structures and holes on the surface after treating the polymer film with enzymatic solutions corresponds to the crystal structure of the films, which is, in turn, highly dependent on the crystallization temperature and tempering conditions.

3.5. Crystallinity Additionally, the polyester films 4 and 5a–e were analyzed by WAXD, as described elsewhere.[19–21] The integration of the reflections in comparison to the amorphous halos results in the degree of crystallinity (Figure 5). A decreasing effect of the aliphatic side chain is visible. This corresponds well to the enthalpy of fusion shown already, which was also decreasing (Figure 2b).

4. Conclusion

Figure 4. Decreasing of remaining mass of polymer films in enzymatic solution for copolymers 4, 5a–e and PCL.


Aliphatic random linear high molecular weight copolyesters with lateral aliphatic chains were obtained by polycondensation of adipic acid, 1,5-pentanediol and 2,2-butyl-ethyl-1,3-propanediol. The introduction of the lateral side chains reduced the crystallinity and, thereby,


211


Table 2. SEM images of polyester films 4, 5a–e and PCL before enzymatic test, after 16 h in pure buffer solution and after 16 h in enzymatic solution.

Polymer

Virgin film

After 16 h in pure buffer solution

After 16 h in enzymatic solution

4

5a

5b

5c

5d

5e

PCL

212




Keywords: degradation; mechanical properties; polycondensation; thermal properties

Figure 5. Decrease of the degree of crystallinity of polymer films 4, 5a–e determined as a ratio of integral of crystalline reflections and complete WAXD diffraction curve.

brittleness of the copolyesters. This property change was accompanied by faster enzymatic degradability, which is of outmost importance for release applications. This set of tunable properties makes aliphatic copolyester an application of major interest in pharmacy, medicine, and agriculture. An important step will be further chemical modifications, for example, by cross-linking the soft copolyesters; this is presently under way in our laboratories.

Acknowledgements: The authors are indebted to the Bundesa€ r Landwirtschaft und Erna €hrung (BLE) for financial gentur fu support.

Received: July 24, 2015; Revised: September 6, 2015; Published online: September 24, 2015; DOI: 10.1002/mabi.201500279


mez-Perez, O. O. Santana, M. L. [1] F. Carrasco, P. Pages, J. Ga Maspoch, Polym. Degrad. Stab. 2010, 95, 116. [2] W. Chumeka, P. Pasetto, J.-F. Pilard, V. Tanrattanakul, J. Appl. Polym. Sci. 2015, DOI: 10.1002/app.41426. [3] N. Grabe, Y. Zhang, S. Agarwal, Macromol. Chem. Phys. 2011, 212, 1327. [4] T. Chen, T. Cai, Q. Jin, J. Ji, e Polym. 2015, 15, 3. [5] S. Philip, T. Keshavarz, I. Roy, J. Chem. Technol. Biotechnol. 2007, 82, 233. [6] R. Reichardt, B. Rieger, in Synthetic Biodegradable Polymers, € nkel, G. W. Coates, R. Reichardt, Vol. 245, B. Rieger, A. Ku E. Dinjus, T. A. Zevaco, Eds., Springer Berlin, Heidelberg 2012. [7] J.-O. Lin, W. Chen, Z. Shen, J. Ling, Macromolecules 2013, 46, 7769. [8] P. Olsen, T. Borke, K. Odelius, A.-C. Albertsson, Biomacromolecules 2013, 14, 2883. [9] H. Ebata, K. Toshima, S. Matsumura, Macromol. Biosci. 2008, 8, 38. [10] N. Kolb, M. A. R. Meier, Green Chem. 2012, 14, 2429. [11] Z. Gan, Q. Liang, J. Zhang, X. Jing, Polym. Degrad. Stab. 1997, 56, 209. [12] Z. Gan, K. Kuwabara, H. Abe, T. Iwata, Y. Doi, Polym. Degrad. Stabil. 2005, 87, 191. [13] H. Cai, V. Dave, R. A. Gross, S. P. McCarthy, J. Polym. Sci. Part B Polym. Phys. 1996, 34, 2701. [14] Y. Ando, K. Yoshikawa, T. Yoshikawa, M. Nishioka, R. Ishioka, Y. Yakabe, Polym. Degrad. Stab. 1998, 61, 129. [15] E. Kun, K. Marossy, Mater. Sci. Forum 2013, 752, 241. [16] M. Nagata, T. Kiyotsukuri, H. Ibuki, N. Tsutsumi, W. Sakai, React. Funct. Polym. 1996, 30, 165. [17] C. G. Pitt, R. W. Hendren, A. Schindler, S. C. Woodward, J. Control. Release 1984, 1, 3. [18] H. Shirahama, Y. Kawaguchi, M. S. Aludin, H. Yasuda, J. Appl. Polym. Sci. 2001, 80, 340. [19] M. Gigli, M. Govoni, N. Lotti, E. D. Giordano, M. Gazzano, A. Munari, RSC Adv. 2014, 4, 32965. [20] M. Kaci, T. Sadoun, S. Cimmino, Int. J. Polym. Anal. Charact. 2006, 6, 455. [21] N. Oleszko, A. Utrata-Wesołek, W. Wałach, M. Libera, ski, B. Trzebicka, A. Dworak, A. Hercog, U. Szeluga, M. Doman Macromolecules 2015, 48, 1852.


213

Microbial degradation of aliphatic and aliphatic-aromatic co-polyesters.

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica.

The effect of mechanical loads on the degradation of aliphatic biodegradable polyesters.

Clickable degradable aliphatic polyesters via copolymerization with alkyne epoxy esters: synthesis and postfunctionalization with organic dyes.

Enzyme-catalyzed synthesis of unsaturated aliphatic polyesters based on green monomers from renewable resources.

Enzymatic synthesis of biobased polyesters using 2,5-bis(hydroxymethyl)furan as the building block.

Shape memory particles capable of controlled geometric and chemical asymmetry made from aliphatic polyesters.

Enzymatic polymerization of cyclic monomers in ionic liquids as a prospective synthesis method for polyesters used in drug delivery systems.

Ambient temperature catalyst-free light-induced preparation of macrocyclic aliphatic polyesters.

Enzymatic degradation of (ligno)cellulose.

P alpha-chiral phosphorothioate analogues of bis(5'-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Degradation mechanisms of bioresorbable polyesters. Part 1. Effects of random scission, end scission and autocatalysis.

Enzymatic synthesis of oligosaccharides.

Enzymatic histamine degradation by human skin.

Facile Enzymatic Synthesis of Ketoses.

Enzymatic synthesis and characterization of galactosyl monoesters.

Sterically hindered malonamide monomers for the step growth synthesis of polyesters and polyamides.

Enzymatic synthesis of magnetic nanoparticles.

Fluorous enzymatic synthesis of phosphatidylinositides.

Enzymatic synthesis of Leu- and Met-enkephalin.

Synthesis and degradation of methylated proteins of mouse organs: correlation with protein synthesis and degradation.

Ring-opening copolymerization (ROCOP): synthesis and properties of polyesters and polycarbonates.

Degradation of recalcitrant aliphatic and aromatic hydrocarbons by a dioxin-degrader Rhodococcus sp. strain p52.

One-pot atom-efficient synthesis of bio-renewable polyesters and cyclic carbonates through tandem catalysis.