Use of thiol-ene click chemistry to modify mechanical and thermal properties of polyhydroxyalkanoates (PHAs)

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Use of thiol-ene click chemistry to modify mechanical and thermal properties of polyhydroxyalkanoates (PHAs) Alex C. Levine a , Graham W. Heberlig a , Christopher T. Nomura a,b,∗ a Department of Chemistry, State University of New York – College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, United States b Center for Applied Microbiology, State University of New York – College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, United States

a r t i c l e

i n f o

Article history: Received 31 July 2015 Received in revised form 16 November 2015 Accepted 18 November 2015 Available online xxx Keywords: Polyhydroxyalkanoates Thiol-ene Biodegradable polymer

a b s t r a c t In order to diversify the number of applications for poly[(R)-3-hydroxyalkanoates] (PHAs), methods must be developed to alter their physical properties so they are not limited to aliphatic polyesters. Recently we developed Escherichia coli LSBJ as a living biocatalyst with the ability to control the repeating unit composition of PHA polymers, including the ability to incorporate unsaturated repeating units into the PHA polymer at specific ratios. The incorporation of repeating units with terminal alkenes in the side chain of the polymer allowed for the production of random PHA copolymers with defined repeating unit ratios that can be chemically modified for the purpose of tailoring the physical properties of these materials beyond what are available in current PHAs. In this study, unsaturated PHA copolymers were chemically modified via thiol-ene click chemistry to contain an assortment of new functional groups, and the mechanical and thermal properties of these materials were measured. Results showed that crosslinking the copolymer resulted in a unique combination of improved strength and pliability and that the addition of polar functional groups increased the tensile strength, Young’s modulus, and hydrophilic profile of the materials. This work demonstrates that unsaturated PHAs can be chemically modified to extend their physical properties to distinguish them from currently available PHA polymers. © 2015 Published by Elsevier B.V.

1. Introduction Of the available biobased and biodegradable polymers, polyhydroxyalkanoates (PHAs) have been well studied as viable alternative to petroleum-based plastics due to their production from renewable resources and inherent biodegradability [1]. PHAs are polyesters that can be natively produced by certain strains of bacteria and have variable physical properties based on the chemical structure of the repeating unit within the polymer. PHAs containing short-chain-length (SCL) repeating units of 3–5 carbon atoms are stiff, crystalline, and brittle, whereas PHAs with mediumchain-length (MCL) repeating units of 6–14 carbon atoms are soft, amorphous, and flexible [2]. In addition to homopolymers of SCL and MCL PHAs, random copolymers have also been produced with both SCL and MCL constituents [3]. Until recently, the inability to

∗ Corresponding author at: Department of Chemistry, State University of New York – College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, United States. E-mail address: [email protected] (C.T. Nomura).

control polymer repeating unit content has resulted in a lack of control over the physical properties in PHA copolymers, thereby limiting the applications for these materials. In particular, the fatty acid substrates used in MCL PHA copolymer production are susceptible to degradation via the ␤-oxidation metabolic pathway, which can alter the length of the fatty acid incorporated into the PHA copolymer, thus resulting in inconsistent and variable physical properties. In particular, the fatty acid substrates used in MCL PHA copolymer production are susceptible to degradation via the ␤-oxidation metabolic pathway, which can alter the length of the fatty acid incorporated into the PHA copolymer, thus resulting in inconsistent and variable physical properties. The physical properties of PHAs are comparable with other polyesters such as polylactic acid [4], polyglycolic acid [5], and polycaprolactone [6]. Polyesters as a class of materials can range from soft and flexible to hard and brittle, with soft polyesters such as polycaprolactone exhibiting a Young’s modulus of 400 MPa [7], and hard polyesters such as polyglycolic acid showing a Young’s modulus of over 12.8 GPa [8]. Melting temperatures of these polyesters can range from 60 to 225 ◦ C, where higher crystallinity correlates with higher melting temperatures [9]. The material

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properties of SCL and MCL PHAs generally fall within the same range, showing melting temperatures of 45–180 ◦ C and tensile modulii of 0.2–3.5 GPa [2]. As an example, the SCL PHA polyhydroxybutyrate (PHB) has high tensile strength and modulus, and a correspondingly low amount of flexibility [10]. Conversely, a MCL PHA such as polyhydroxyoctanoate has low strength but exhibits increased flexibility and high elongation to break [11]. Blends and copolymers of PHAs allow for the production of a range of physical properties from SCL to MCL homopolymers. Poly(hydroxybutyrate)-co-(hydroxyvalerate) (PHBV) is a SCL PHA copolymer that has been extensively studied [10,12–15] due to its balance of high tensile strength flexibility, where increasing the PHV content decreases the crystallinity of the material leading to reduced brittleness [16]. Recently, we engineered a strain of Escherichia coli with the ability to control both the identity and quantity of repeating units in PHA homopolymers and copolymers [17,18]. This was important for not only reproducibility and control over the polymer’s physical properties, but also because it allowed for the production of novel PHA homopolymers and copolymers. In native PHA producing bacteria, fatty acid substrates that can be used for PHA production are subject to reduction in chain length from metabolic processes such as ␤-oxidation. This leads to an unequal distribution of repeating units and an inability to precisely control the size of the repeating units within the PHA polymer. E. coli LSBJ, the engineered strain of bacteria used in this study, can convert fatty acids to enoylCoA substrates that bear the same number of carbon atoms as the fatty acid feedstock fed to it. This ability, along with the deletion of key enzymes in the ␤-oxidation pathway, have allowed for the production of PHA homo- and copolymers with PHA compositions derived solely from fatty acids present in the growth medium. Despite the fact that PHA polymers may be inherently suitable as substitutes for petroleum-based plastics, applications have generally been limited to bulk-commodity plastics replacements [19,20]. Much of this limitation has been due to a lack of chemical reactivity in the non-modifiable aliphatic hydrocarbon side chains in the repeating units of PHA polymers. It was hypothesized that by modifying the side-chain of PHA polymers to contain functional groups other than alkyl chains, the physical properties of the polymer could be modulated based on the identity of the new functional group. In this study, we demonstrate the production of chemically modifiable PHA polymers and their subsequent chemical modification to expand and improve upon material properties. There have been previous efforts made to bacterially produce unnatural PHAs [21,22] or chemically modify PHA polymers in order to improve their physical properties [23–27]. Past studies have focused on using PHAs with unsaturated side chains as a means of altering their properties [28,29]. In particular, it has been recently shown that the terminal alkene functional group of unsaturated PHA copolymers can be modified [30,31], resulting in changes to the polymer’s properties. One study investigated the synthesis of a water soluble PHA [32] through a two-step reaction involving epoxidation of the alkene followed by the addition of diethanolamine, and this material was further tested for applications in DNA plasmid delivery [33]. Other studies have used thiol-ene click chemistry to modify unsaturated PHAs for use in amphiphilic graft copolymers [34]. The work presented here uses thiol-ene click chemistry [35,36] as a means to alter the thermal and mechanical properties of unsaturated PHAs via side-chain modification of their alkene functional group. For the purpose of this study, the incorporation of three types of functional groups into the side-chain of the PHA polymer were assessed, including alcohol, acid, or cross-linkable ester function groups, and the resulting changes in properties were measured. Relevant characteristics such thermal properties, tensile strength, Young’s modulus, and hydrophobicity were

measured and compared to unmodified PHA. Results of this work provide evidence that chemical modification can be used to modify PHA polymers to improve strength and pliability. 2. Materials and methods 2.1. Production of poly[(R)-3-hydroxybutyrate-co-(R)3-hydroxy-10-undecenoate] (PHBU) using E. coli LSBJ The SCL/MCL copolymer PHBU was produced in a manner similar to that described for the production of poly[(R)-3hydroxybutyrate-co-(R)-3-hydroxyoctanoate] by Tappel et al. [18]. The strain used for polymer production was E. coli LSBJ, a derivative of E. coli LS5218 with deletion of genes fadB and fadJ, and harboring a plasmid containing the genes for an (R)-specific enoyl-CoA hydratase (phaJ4) and PHA synthase [phaC1(STQK)]. Starter cultures consisted of a 250 mL Erlenmeyer flask containing 50 mL Lennox broth (composition per liter: 10 g tryptone, 5 g yeast extract, and 5 g sodium chloride) and 50 mg/L kanamycin, which was inoculated with E. coli LSBJ and incubated for 16–18 h at 30 ◦ C in an orbital shaker set at 250 rpm. From this starter culture, 20 mL were taken as an inoculum for the subsequent fermentation. Fermentations were conducted using a New Brunswick Scientific Bioflo 310 Fermentor/Bioreactor equipped with a 5 L fermentation vessel (New Brunswick Q479700). For the standard curve of PHBU production with different repeating unit compositions, the growth medium remained unchanged except for the ratio of fatty acids added. Fermentations consisted of 2 L of LB culture medium supplemented with 2 g/L of fatty acids sodium butyrate and 10-undecenoic acid (Alfa Aesar), 4 g/L Brij-35 (Fisher Scientific), and 50 mg/L kanamycin. The role of Brij-35 in these experiments was as a surfactant to aid in the solubilization of 10-undecenoic acid, which was insoluble in aqueous medium. Without complete solubility, it is unlikely that the MCL fatty acid would be taken up by the bacterium for PHA production at rate comparable to the fatty acid salt sodium butyrate. The fermentation parameters were agitation at a constant 300 rpm, air flow maintained at 0.5 standard liters per minute (SLPM), temperature held at 30 ◦ C, and the duration of the fermentation lasted for 48 h after inoculation. Following fermentation, cells were harvested with centrifugation for 15 min at 3716 × g and 22 ◦ C, and the supernatant discarded. Cell pellets were washed with 70% ethanol (Fisher Scientific) to remove residual fatty acids, collected again by centrifugation under the same conditions, and washed with nanopure water. After washing with water, the cells were collected by centrifugation once more and resuspended in 15 mL of water before freeze drying. After drying by lyophilization, the cells were suspended in methanol (22 mL/g dried cell mass, Fisher Scientific) and gently stirred for 5 min at 22 ◦ C. Afterwards, cells were collected by centrifugation and washed with nanopure water. Cells were again dried by lyophilization, and PHA polymers were purified via Soxhlet extraction followed by methanol precipitation. Soxhlet extractions were performed in 120 mL of chloroform (Sigma–Aldrich) for 5 h, and then the solutions were transferred to a glass petri dish where the solvent was evaporated under ambient conditions for >6 h. The resulting films were dissolved in a minimal amount of chloroform, and precipitated into a 10-fold larger volume of methanol. After filtration, the polymer was dried under vacuum at 25 ◦ C for 48 h and stored at 22 ◦ C in the dark. 2.2. Molecular weight determination of PHA polymers Molecular weights of polymers were determined by gel permeation chromatography (GPC) as previously described [37]. Samples

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Scheme 1. Chemical modification of PHBU with alcohol, acid, or cross-linked functional groups.

were prepared for GPC by transferring 1–2 mg of polymer to a 5 mL glass vial and were dissolved in chloroform to a final concentration of 0.7 mg/mL by heating at 50 ◦ C. Once dissolved, 1 mL of solution was filtered through a 0.45 ␮m PTFE syringe filter into a 2 mL GPC vial. Samples volumes of 50 ␮L were injected into a LC-20AD liquid chromatograph equipped with a SIL-20A autosampler, CTO-20A column oven, and an RID-10A refractive index detector (Shimadzu). Chromatography was performed using a 8 mm × 50 mm styrenedivinylbenzene (SDV) guard column (5 ␮m particles; Polymer Standards Service) and a 8 mm × 300 mm SDV analytical column (5 ␮m particles; mixed bed porosity; max molecular weight 1E6 Da; Polymer Standards Service product sda083005lim). The mobile phase consisted of chloroform at a flow rate of 1 mL/min and the temperature was maintained at 40 ◦ C. Analysis was performed using Shimadzu LCsolution software. 2.3. Synthesis of chemically modified PHBU PHBU was modified with either 2-mercaptoethanol or 3mercaptopropanoic acid to produce alcohol or acid pendant functional groups, respectively (Scheme 1). To a 20 mL glass scintillation vial, 0.10 g of PHBU (8.0 × 10−5 moles of alkene) was added and dissolved in 5 mL of anhydrous tetrahydrofuran (THF) (Sigma–Aldrich) by incubating at 55 ◦ C for 1 h. To the polymer solution, either 8.0 × 10−5 moles (6.3 ␮L) of 2-mercaptoethanol (Alfa Aesar) or 8.0 × 10−5 moles (8.5 ␮L) of 3-mercaptopropanoic acid (MPA) (Alfa Aesar) was added along with 1 mg of radical initiator 2,2-dimethoxy-2phenylacetophenone (DMPA) (Sigma–Aldrich). The reactions were irradiated with 365 nm UV light using a UVP Black-Ray® XX-15L UV Bench Lamp for 30 min at 20–22 ◦ C. To confirm the product, a 1 mL portion of the reaction was collected and dried under vacuum at 25 ◦ C for 48 h, then analyzed by NMR spectroscopy. 2.4. Synthesis of cross-linked PHBU To a vial containing 0.10 g of PHBU (8.0 × 10−5 moles of alkene), 5 mL of anhydrous THF was added and the polymer dissolved by incubating at 55 ◦ C with stirring for 1 h. Following, an amount of pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) (Sigma–Aldrich) was added corresponding to the desired amount of cross-linking, including equivalents of 0.25, 0.50, 0.75, and 1.00 (2.0 × 10−5 , 4.0 × 10−5 , 6.0 × 10−5 , and 8.0 × 10−5 moles of alkene, respectively). Actual additions of PETMP were 2, 4, 8, and 16 ␮L, respectively. Additionally, 1 mg of DMPA was added, and the solution was irradiated with 365 nm UV light for 30 min at 20–22 ◦ C

to induce cross-linking. Films were cast by allowing the solvent to evaporate under vacuum at 25 ◦ C for 48 h. 2.5. Characterization of modified PHBU by 1 H NMR The repeating unit content of PHBU was determined by 1 H NMR spectroscopy using a Bruker AVANCE 600 spectrometer. Samples consisted of 10–15 mg of polymer dissolved in 1 mL of deuterated chloroform. Spectra were processed using TOSPIN v1.3 from Bruker BioSpin. The ratio of the alkene proton signal at 5.8 ppm to the polymer backbone stereocenter at 5.2 ppm was taken as the MCL portion of the copolymer. A series of PHBU copolymers with varying MCL content were generated to produce a standard curve for precise PHBU synthesis. From this data, a PHBU copolymer with 8% MCL was produced. The alcohol and acid modified PHBU products were also confirmed by 1 H NMR spectroscopy (Bruker AVANCE 600 spectrometer). Samples consisted of 15 mg of polymer dissolved in 1 mL of deuterated chloroform. Spectra were processed using TOSPIN v1.3 (from Bruker BioSpin) based on the disappearance of the alkene proton signals at 5.0 and 5.8 ppm. The disappearance of these signals indicated that the alkene function group had been converted to the thioether product. 2.6. Characterization of cross-linked PHBU by FT-IR Due to the insolubility of cross-linked PHBU, 1 H NMR spectroscopy was not appropriate for chemical characterization. The conversion of alkene functional groups to thioether cross-links was instead measured by FT-IR, where the decrease in the alkene signal at 1641 cm−1 was taken as evidence of successful reaction and approximate amount of cross-linking as previously described [38]. Samples were analyzed using a Bruker Tensor 27 spectrometer equipped with an attenuated total reflection (ATR) stage. Data collection was performed using OPUS 6.5 software, and each spectrum consisted of 32 scans plotted in transmittance mode. Data was re-plotted using Microsoft Excel 2007 to generate higher quality images. 2.7. Analysis of thermal and physical properties Stress/strain curves were generated for each sample using a TA Q800 DMA. Samples were prepared via solvent casting for alcohol and acid modified polymers, and in the case of cross-linked samples which resulted in gels, the reactions were performed in the same glass vessel used for casting to maintain shape. After film formation, samples were dried under vacuum at 25 ◦ C for 72 h to remove solvent. It is known that residual amounts of solvent can

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have a plasticizing effect in polymers [39], which makes the solvent removal step critical for accurate analysis of physical properties. Post-manufacture aging and storage conditions were 2–5 days and 20–22 ◦ C in a dry environment. Films were cut into 2 mm × 15 mm strips, with thicknesses ranging from 0.15 to 0.25 mm, and clamped into the instrument with the aid of a 5.00 mm spacer to ensure reproducibility. Each sample was pulled at a constant rate of 2.00 mm/min until break, at ambient temperature (20–22 ◦ C). The initial slope of the curve was taken as the First modulus (Young’s modulus), and the slope of the cross-linked portion of the curve was taken as the Second modulus (cross-link modulus), when applicable. The tensile strength of each sample was taken at the highest point in the curve, appearing just before break. Thermal property data was collected using a TA Q200 DSC in Heat/Cool/Heat mode, where samples were heated at a rate of 10 ◦ C/min up to 250 ◦ C, cooled down to −80 ◦ C at a rate of 5 ◦ C/min, and re-heated to 250 ◦ C at a rate of 10 ◦ C/min. The initial heat ramp was used to erase any thermal history of the sample, with glass transition temperature (Tg ), crystallization temperature (Tc ), and melting temperature (Tm ) values taken from the second heating ramp. Degradation temperature (Td ) was obtained using a TA Q5000 TGA with a heat ramp from 30 ◦ C to 250 ◦ C at a rate of 10 ◦ C/min. All thermal and physical property data was collected using TA Universal Analysis software.

10-undecenoic acid in polymer (%)

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100 80 60 40

y = 0.927x + 2.532 R² = 0.986

20 0 0

20

40

60

80

100

10-undecenoic acid in medium (%) Fig. 1. Standard curve of PHBU copolymer production in E. coli LSBJ. Unsaturated repeating unit content was expressed as a percentage of total repeating units in the copolymer.

2.8. Water contact angle Static contact angle measurements of PHA films were conducted using a Rame-Hart 250-F1 standard goniometer. Prior to contact angle measurements, films were cast from 0.10 g of polymer dissolved in 10 mL of THF. Films were dried under vacuum at 25 ◦ C for 48 h to remove residual solvent. For each sample, a droplet of water was placed on the flat surface of the film and the contact angle was immediately measured using DROPimage Advanced software. 3. Results 3.1. Production and characterization of PHBU copolymer A PHA copolymer was designed with unsaturated repeating unit content that could be chemically modified for either cross-linking or side-chain modification with polar functional groups. We chose to produce a PHA copolymer with sufficient MCL repeating unit content for alkene modification and elasticity and sufficient SCL content for thermoplastic strength. To meet these material property requirements, a SCL–MCL PHA copolymer with 8% MCL content and 92% SCL content was produced for its balanced physical properties and its ample amount of MCL content for modification as described previously [17]. The fatty acids chosen as precursors for polymer production were butyric acid (sodium butyrate) and 10undecenoic acid. Fermentation of E. coli LSBJ with these feed stocks resulted in the production of the poly[(R)-3-hydroxybutyrate-co(R)-3-hydroxy-10-undecenoate] (PHBU) copolymer. A series of PHBU copolymers were produced in a manner described previously [38], but scaled for production in a fermenter as opposed to shake flasks. PHA copolymers with a range of MCL repeating unit content were produced in order to determine the parameters to make copolymers with a final desired ratio of repeating units (Fig. 1). The repeating unit contents of PHBU were determined by 1 H NMR spectroscopy (Fig. 2), where the ratio of the alkene proton signal at 5.8 ppm to the polymer backbone stereocenter at 5.2 ppm was taken as the MCL portion of the copolymer. The yield of PHBU was approximately 50 wt% of the cell dry weight, which was similar to previous studies. The polymer molecular weight data determined via GPC is represented in Table 1. The weight average molecular weight (Mw ) was

Fig. 2. 1 H NMR spectrum of a PHBU copolymer containing 8% unsaturated repeating units, as calculated by the integration of peak “h”.

estimated to be 356 kDa, and the number average molecular weight (Mn ) of PHBU in was estimated to be 74.3 kDa. Mn was used in the stoichiometric calculations for chemical modification. 3.2. Synthesis of modified PHBU Two distinct modifications were explored to modify the PHBU polymer. Using thiol-ene click chemistry, an alcohol functional group was introduced into the side-chain of the polymer via addition of 2-mercaptoethanol, and an acid functional group was added using 3-mercaptopropanoic acid, respectively. As expected, the addition of these functional groups into the side-chain of the polymer via thiol-ene click chemistry resulted in a high yield of the modified PHBU product. Based on 1 H NMR spectroscopy, the yield of PHBU-OH was greater than 95% (Fig. 3), and the yield of PHBUCOOH was at least 88% (Fig. 4). These yields were determined based on the integrations of the alkene proton signals at 4.9 ppm and 5.8 ppm, where larger reductions in these signals corresponded to higher yields of product.

Table 1 Molecular weight of produced PHBU copolymers. Sample (%PHU)

Method

Mw (kDa)

Mn (kDa)

Mw /Mn

PHBU8 PHBU36 PHBU65

5 L fermentor 5 L fermentor 5 L fermentor

356 ± 51.7 237 ± 37.2 172 ± 31.8

104 ± 12.7 76.1 ± 10.0 59.5 ± 11.7

3.4 ± 0.1 3.1 ± 0.1 2.9 ± 0.1

Mw , weight average molecular weight; MN , number average molecular weight; Mw /MN , polydispersity; kDa, kilodaltons; PHBU, poly[(R)-3-hydroxybutyrateco-(R)-3-hydroxy-10-undecenoate]; %PHU, poly[(R)-3-hydroxy-10-undecenoate] percentage in copolymer.

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PHBU PHBUX 0.25 PHBUX 0.50 PHBUX 0.75 PHBUX 1.00

1660

1650

1640

1630

1620

Wavelength (cm-1)

Fig. 3. 1 H NMR spectrum of PHBU after thiol-ene click reaction with betamercaptoethanol. This spectrum dictated a high conversion of PHBU to the PHBU-OH product based the on reduction of the alkene peak at 5.8 ppm.

Fig. 5. FT-IR spectra of cross-linked PHBU polymers. The extent of cross-linking was controlled by concentration of thiol cross-linker, as evident by the decrease of the alkene signal at 1641 cm−1 .

3.4. Water contact angle 3.3. Cross-linked PHBU The third type of chemical modification to PHBU was a cross-linking reaction. Using the tetra-thiol PETMP, a series of cross-linked PHBU were produced with increasing amounts of functionalization. Four cross-linked samples were produced by varying the concentration of the PETMP cross-linker in each of the reactions. The samples were designated PHBUX0.25 , PHBUX0.50 , PHBUX0.75 , and PHBUX1.00 , which correspond to the mole equivalent of thiols in the PETMP cross-linker that were added to each reaction, with respect the moles of alkene present from the PHBU polymer. In this scenario, PHBUX1.00 contains an equimolar amount of thiol and alkene functional groups, representing a maximum possible amount of modification. In every cross-linked sample, gellation occurred after 30 s of irradiation of UV light regardless of PETMP concentration, indicating that the cross-linking reaction was rapid. Due to the insolubility of the cross-linked polymers, 1 H NMR could not be used to assess the yield of the reactions. Instead, FT-IR spectroscopy was used to qualitatively determine the conversion of the alkene functional groups in the cross-linked product. Similar to the monitoring of alkene proton signal reduction in PHBU-OH and PHBU-COOH, the reduction of the alkene signal in the FT-IR spectrum at 1641 cm−1 was taken as evidence for the conversion of the alkene functional group to the thioether product. FT-IR data showed that increasing the concentration of PETMP correlated to a further decrease in the alkene signal, which was nearly absent in the PHBUX1.00 sample (Fig. 5).

Water contact angle was used to assess the change in surface hydrophillicity in the modified PHBU-OH and PHBU-COOH samples. Results showed that contact angle decreased in both instances of modification, where the PHBU contact angle of 81.2◦ was decreased to 73.1◦ for the alcohol modification (Fig. 6A), and 74.8◦ for the acid modification (Fig. 6B). 3.5. Thermal property analysis of modified PHBU The changes in thermal properties of the modified polymers were determined using DSC and TGA. The degradation temperature of the modified samples was increased in every case, from 288.2 ◦ C in the unmodified PHBU to as high as 301.1 ◦ C in the crosslinked PHBUX1.00 (Table 2). Changes were also observed in the glass transition temperatures, which were increased in some samples, but decreased in others (PHBUX0.25 and PHBUX0.75 ). Changes to crystallization temperature varied based on the type of modification. PHBU-OH showed a slight increase in Tc from 74.4 ◦ C in the unmodified PHBU to 76.9 ◦ C, but there was no detectable Tc in the acid modified PHBU-COOH. The three cross-linked materials with the lowest cross-link concentrations showed reduced Tc from 74.4 ◦ C down to 68.8 ◦ C or less, however the sample with the highest amount of cross-link, PHBU1.00 , showed an increase in Tc to 80.9 ◦ C. Melting temperatures were decidedly lower in all modified polymers, with the sharpest decrease seen in PHBUX0.75 from 143.2 ◦ C to 116.4 ◦ C. 3.6. Mechanical property analysis of modified PHBU To assess changes in the mechanical properties of the modified polymers, stress/strain curves were generated to determine tensile strength, elongation, and Young’s modulus (Fig. 7). All cross-linked samples showed significant increases in elongation to break and tensile strength. Elongation was increased to 495.2% in PHBUX0.25

Table 2 Thermal property data of modified PHBU.

Fig. 4. 1 H NMR spectrum of PHBU after thiol-ene click reaction with mercaptopropionic acid. The spectrum indicated high levels of conversion of the PHBU to the PHBU-COOH product based the on reduction of the alkene peak at 5.8 ppm.

Sample

Td (◦ C)

Tg (◦ C)

Tc (◦ C)

Tm (◦ C)

PHBU PHBU-OH PHBU-COOH PHBU-X 0.25 PHBU-X 0.50 PHBU-X 0.75 PHBU-X 1.00

288.2 289.7 287.7 296.9 296.0 298.6 301.1

−9.6 −8.9 −7.5 −13.2 −8.1 −16.8 −8.4

74.4 76.9 N.D. 68.8 66.8 67.8 80.9

143.2 134.0 123.7 124.2 130.0 116.4 132.0

Td , degradation; Tg , glass transition; Tc , crystallization; Tm , melting; N.D., not detected.

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Fig. 6. Water contact angle of PHBU (A) and PHBU-COOH (B). Chemical modification with alcohol functional groups (PHBU-OH) (73.1◦ ) and acid functional groups (PHBUCOOH) (74.8◦ ) both resulted in decreased contact angles from the original PHBU (81.2◦ ).

Table 3 Mechanical property data of modified PHBU. Sample

Elongation (%)

PHBU PHBU-OH PHBU-COOH PHBUX 0.25 PHBUX 0.5 PHBUX 0.75 PHBUX 1.00

88.8 28.0 67.4 495.2 444.7 340.1 280.4

± ± ± ± ± ± ±

Young’s modulus (MPa)

19.2 3.9 31.9 67.1 13.9 9.6 20.3

367.5 605.0 527.4 334.9 366.6 372.9 274.2

± ± ± ± ± ± ±

43.8 70.7 33.6 13.4 32.1 44.3 66.4

Cross-link modulus (MPa)

Tensile strength (MPa)

N.D N.D. N.D. 3.2 ± 0.4 3.8 ± 0.3 6.0 ± 0.4 4.1 ± 0.3

8.5 11.3 11.6 20.0 20.4 23.0 16.4

± ± ± ± ± ± ±

0.5 1.0 0.8 2.1 1.3 0.7 0.5

MPa, megapascals; N.D., not detected.

15

Stress (MPa)

12 9 PHBU PHBU-OH

6

PHBU-COOH PHBU-X 0.25

3

PHBU-X 0.50 PHBU-X 0.75

0

The changes in mechanical properties for alcohol modified PHBU-OH and acid modified PHBU-COOH were significantly different than changes resulting from cross-linking. There was a decrease in elongation from 88.8% in PHBU to 67.4% in PHBU-COOH and 28.0% in PHBU-OH. Young’s modulus was significantly increased from 367.5 MPa in PHBU to 527.4 MPa in PHBU-COOH and 605.0 MPa in PHBU-OH. Tensile strength was also increased from 8.5 MPa in PHBU to 11.6 MPa in PHBU-COOH and 11.3 MPa in PHBU-OH.

0

20

40

60

80

100

Strain (%) Fig. 7. Stress–strain curves of PHBU and modified PHBU. Significant changes to tensile strength (stress) and elongation to break (strain) were observed, and varied depending on the type of modification. *Data for samples PHBU-X 0.25, PHBU-X 0.50, and PHBU-X 0.75 have abbreviated strain values in order to show comparisons with lower strain samples. Full stress strain curves for these materials can be found as supplemental data.

from 88.8% in PHBU, but trended downward with increasing amounts of cross-linking to 280.4% in PHBUX1.00 (Table 3). The tensile strength of the cross-linked samples increased with increasing amounts of functionalization from 8.5 MPa in PHBU to 23.0 MPa in PHBUX0.75 , but a drop in tensile strength was observed for PHBU1.00 to 16.4 MPa. Young’s modulus was not significantly different between PHBU, PHBUX0.25 , PHBUX0.50 , and PHBUX0.75 , however a sharp decrease was seen in PHBUX1.00 to 274.2 MPa from 367.5 MPa in PHBU. Interestingly, the shape of the stress strain curves was different in cross-linked samples as compared to unmodified PHBU. In the region of plastic deformation, the cross-linked samples showed a linear increase in tensile strength as compared to virtually no increase in the PHBU sample (see Supplementary data Figs. S1–S7 for complete stress–strain curves). In these regions of linear increase in tensile strength, the slope was taken and designated as a “cross-link” modulus to compared the modulus of the cross-linked samples in their regions of deformation. These crosslink modulii increased with increasing amounts of cross-linking, from 3.2 MPa in PHBUX0.25 to 6.0 MPa in PHBUX0.75 . This cross-link modulus decreased in PHBUX1.00 to 4.1 MPa.

4. Discussion One of the aims of this work was to produce PHAs with improved physical properties such as increased tensile strength. While it would be possible to produce unsaturated PHA polymers from native PHA producing bacteria, these native producers would not produce a polymer with only the two intended repeating units (4 carbon and 11 carbon), but would produce a single random copolymer containing many repeating unit identities due to the degradation of the 11-carbon fatty acid substrate via ␤-oxidation (resulting in repeating units of 9, 7, and 5 carbons, for example). As has been studied previously, the uncontrolled nature of repeating unit identities in the PHA polymers produced in native bacterial strains result in highly variable physical properties. In order to accurately measure improvements to the physical properties of the chemically modified PHA polymers produced in this study, it was prudent to reduce the variability as much as possible. This is the reason why PHA production was done in E. coli LSBJ, in order to produce a copolymer with only two repeating unit identities, thus reducing the variation in physical properties typically observed in polymers produced from native PHA producing strains. In an effort to expand the properties of PHA for a broader set of applications beyond petroleum-based, bulk-commodity plastics replacements, unsaturated PHA polymers were produced with defined quantities of alkene functional groups to act as chemical handles for synthetic chemical modification. The PHBU polymer produced in this study had the same chemical composition as found in a previous study [38], as it was determined in other works [17] that a SCL–MCL PHA copolymer with 5–10% MCL content exhibited the best balance of strength and flexibility. Thiol-ene click chemistry was found to be an efficient reaction to modify PHBU. With reaction times of 30 min, stoichiometric amounts of reagent, and

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ambient temperatures, this reaction was able to convert >85% of the available alkenes to thioether product. Cross-linking reactions have shown great potential to improve the physical properties of polymers [40–43]. For cross-linked samples, it was evident that new bonds were formed rapidly due to the onset of gellation, which occurred in seconds. When analyzed by FT-IR, it was found that the conversion of reactant alkene functional groups to the thioether product correlated with thiol concentration. With the purpose of tailoring the physical properties of PHA to match currently used synthetic plastics, this method based on thiol-ene click chemistry allows for the control over cross-link concentration, and thus stringent control over cross-linking, at the level of reactant concentration. By changing this one parameter, as opposed to needing excess reactant, time, or temperature, the goal of strengthening the polymer can be accomplished, and this result should translate directly to larger sample sizes closer to commercial application [19]. Of the changes in thermal properties, the most significant result was the change in polymer melting temperature. With modification of the polymer drops in melting temperature of at least 10 ◦ C were seen, with one sample showing a drop from 143.2 ◦ C in the unmodified PHBU to 116.4 ◦ C in the cross-linked PHBUX0.75 . Decreases in melting temperature are important for processes such as compression molding [13], where polymers are heat pressed into shape for a desired application. This drop in melting temperature brings cross-linked PHBU into the same range as high density polyethylene (115–135 ◦ C) [17]. While the changes in thermal properties were mild, improvements in mechanical properties were significant for the chemically modified PHBU copolymers. Compared with petroleum-based plastics or other polyesters [44–47], SCL–MCL PHAs are limited by low deformation potential. Results showed that cross-linking the PHBU polymer increased the elongation to break values from 88.8% in unmodified PHBU to 495.2% in PHBUX0.25 . This increase in pliability represents a 450% improvement, which was coupled with an improvement in tensile strength of 20.0 MPa, a 135% improvement over the unmodified PHBU at 8.5 MPa. These changes together describe a material that is very close in thermal and physical properties to polyethylene, but due to its polyester backbone, will exhibit far superior biodegradability compared to strictly hydrocarbon polymers. The change in shape of the stress/strain curve for cross-linked PHBU polymers to a J-shape curve [48] allowed for the measurement of a modulus separate from the traditional Young’s modulus. Whereas the initial slope of the stress/strain curve is typically taken as the Young’s modulus [49], representing the resistance of the material to deformation, cross-linked PHBU showed a second region of linear increase in tensile strength, and the slope of this region was taken as a cross-link modulus. This value represents the resistance to deformation as a result of the intra/intermolecular cross-links in the polymer. Values of cross-link modulus ranged from 3.2 MPa to 6.0 MPa, and a clear trend was seen comparing this cross-link modulus to properties such as elongation and tensile strength. This parameter would be important in applications interested in deformation values at high elongation or maximum obtainable tensile strength [50]. An important parameter for PHA biodegradability is hydrophobicity. Considering the major routes of PHA biodegradation, hydrolytic cleave of the polyester backbone and enzymatic degradation [3], the ability to wet the surface of the polymer is critical to control rates of degradation. The water contact angle of alcohol and acid modified PHBU were examined to determine whether these polymers exhibited increased surface hydrophilicity, which could affect rates of biodegradation. The change in contact angle was moderate, decreasing by 6.4◦ in acid modified PHBU and 8.1◦ in alcohol modified PHBU. Further improvements to contact angle

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could be achieved by increasing the alkene content in the PHA polymer, as this would allow for greater amounts of functionalization. The PHBU used in this study with 8% of its repeating unit content containing the alkene functional group was chosen due to its inherent balance of physical properties, which in turn limits the amount of possible modification by polar functional groups. If a PHA with higher surface wetting capability were desired, a different PHBU copolymer with higher alkene content and subsequent modification could see improved results. This type of PHA may also find use in tissue-engineering applications where surface hydrophilicity is important for cell attachment [51,52]. 5. Conclusion The objective of this work, the expansion of available physical properties in PHA polymers was accomplished through the chemical modification of an unsaturated random PHA copolymer via thiol-ene click chemistry. Following production of unsaturated PHBU, the polymer was successfully modified to contain side-chain alcohol, acid, or cross-linked functional groups, and these moieties were able to provide new combinations of properties not available in current poly([R]-3-hydroxyalkanoates). Previously, the constraints on the physical properties of SCL–MCL PHA copolymers were limited to either high tensile strengths or high elongation to break values [17]. The work shown here has outlined a method to produce PHA copolymers with both high strength and flexibility through cross-linking. Other modifications were shown to improve tensile strength, Young’s modulus, and hydrophilicity with the incorporation of polar functional groups. The chemical modifications outlined in this work illustrate the potential for an array of new PHA polymers with previously unavailable combinations of properties. The ability to chemically modify PHA polymers postproduction, which is absent in common aliphatic petroleum-based plastics, will afford new strategies to tailor PHAs for functions beyond current applications. Author contributions The manuscript was written by Alex C. Levine and Christopher T. Nomura. Experiments were designed by Alex C. Levine and Christopher T. Nomura. Graham W. Heberlig assisted in PHA production. Alex C. Levine carried out all other experiments. Acknowledgements This study was made possible by an NSF CBET award (1263905) to C.T. Nomura. We also acknowledge the SUNY-ESF Honors Program for support of this project. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2015. 11.048. References [1] J. Lu, R.C. Tappel, C.T. Nomura, Mini-review: biosynthesis of poly(hydroxyalkanoates), Polym. Rev. 49 (2009) 226–248. [2] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, Prog. Polym. Sci. 25 (2000) 1503–1555. [3] A.P. Bonartsev, V.L. Myshkina, D.A. Nikolaeva, E.K. Furina, T.A. Makhina, Biosynthesis, biodegradation, and application of poly (3-hydroxybutyrate) and its copolymers – natural polyesters produced by diazotrophic bacteria, Commun. Curr. Res. Educ. Top. Trends Appl. Microbiol. (2007) 295–307.

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