Simple synthesis of amphiphilic poly(3-hydroxy alkanoate)s with pendant hydroxyl and carboxylic groups via thiol-ene photo click reactions

(1)

Simple synthesis of amphiphilic poly(3-hydroxy alkanoate)s with

pendant hydroxyl and carboxylic groups via thiol-ene photo click

reactions

*

Baki Hazer

*

Bülent Ecevit University, Department of Chemistry, 67100 Zonguldak, Turkey

a r t i c l e i n f o

Article history:

Received 16 February 2015 Received in revised form 5 April 2015

Accepted 27 April 2015 Available online 7 May 2015 Keywords:

Unsaturated PHAs

Thiol-ene photo click reaction 3-Thio glycerol

Mercapto propionic acid HMBC-NMR technique

a b s t r a c t

Biodegradable polymers gained worldwide attention among researchers because of environmental and petroleum reserve limitation issues. In this manner, poly (3-hydroxyalkanoate)s, PHAs, are very useful materials from the point of this view. They can be easily obtained by using several bacteria from renewable substrate such as sugar, plant oils and as well as synthetic chemicals. The improvement of their mechanical properties and enhance hydrophilic character are still main challenge of the polymer scientists. Herein we report the thiol-ene photo click reactions of the unsaturated medium chain length PHAs produced by using Pseudomonas oleovorans from 10-undecenoic acid, octanoic acid and/or soybean oily acids that are coded as poly(3-hydroxy undecenoate) (PHU), poly(3-hydroxy octanoate-co-undecenoate) (PHOU) and poly(3-hydroxy octanoate-co-soybean oil polymer) (PHOSy), respectively, in order to obtain their hydroxyl and carboxyl derivatives. The molecular weights of the modiﬁed PHAs obtained in this work were the same as those of the starting PHAs. Structural analysis of the PHA de-rivatives was performed by using 1H-, 13C, HMBC and HSQC NMR techniques. Melting and glass tran-sitions of the hydroxyl and carboxyl derivatives of the microbial polyesters were found to be relatively higher than that of the starting unsaturated PHAs.

1. Introduction

Biodegradable polymers have attracted very high attention by the governments in the world because of environmental and pe-troleum reserve limitation issues[1e4].

PHAs are very useful materials from the point of this view. They can be easily obtained via renewable substrate such as sugar and plant oils from several bacteria through fermentation process [5e16]. However, their mechanical properties are needed to improve in the view of the elongation. At the same time, their high hydrophobic character limits their use in drug delivery systems. To improve their mechanical properties and hydrophilic character are still main challenge for the polymer scientists[17e27]. Over the last two decades, lots of results have been reported in scientiﬁc area but very little was reﬂected to industry. When plant oils and 10-undecenoic acids are used as substrates, unsaturated

poly-3-hydroxy alkanoates, which are highly open for chemical modi fica-tion reacfica-tions, are obtained[28e31]. Pendant unsaturated moieties of the unsaturated PHAs can be transformed to epoxide groups by the chemical modification reactions of double bonds with per-benzoic acid[32,33]. Epoxidized PHAs can be transformed to al-cohols and carboxylic acids by using potassium permanganate [34,35], Osmium tetroxide[36], and 9-borobicyclononane in or-der to obtain amphiphilic PHAs with hydroxyl and carboxylic acid groups[37]. Sparks and Scholz synthesized the cationic PHA by the reaction of pendent epoxidized double bonds with diethanol amine [38]. We recently reported their chloride derivatives[39]and some graft copolymers with poly (methyl methacrylate) and polystyrene [40,41]. Cross-linking of the unsaturated PHAs was also successfully achived[42e44].The well-known thiol-ene click reaction[45e52] was also used to prepare PHOU-g-polyethylene glycol amphi-philic graft copolymers in order to synthesis multi compartment micelles[53,54]. In this manner, the modified PHAs with pendant grafts via thiol-ene click reactions by using 2-per fluorooctyl-1-ethanethiol and PEG 550eSH in the presence of 2, 2’-azo-bis iso-butyronitrile was accomplished.

*_{This work is dedicated to late Prof. Dr. Robert (Bob) W. Lenz.} * Tel.: þ90 372 2911372; fax: þ90 372 2914181.

E-mail address:bkhazer@beun.edu.tr.

Contents lists available atScienceDirect

Polymer Degradation and Stability

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p o l y d e g s t a b

http://dx.doi.org/10.1016/j.polymdegradstab.2015.04.024

(2)

The PHAs with enhanced hydrophilicity and improved me-chanical properties make this material very good candidates for the applications both in industry and medicine[55e57]. Therefore, the modiﬁcation reactions of the PHAs are still very attractive research area for polymer scientists. In terms of thiol-ene photo click re-actions, benzophenone is a type of photoinitiator, which can

generate active free radicals through photo cleavage and is widely used for photo induced polymerization of vinyl monomers. Benzophenone cannot be used as a photoinitiator in visible light because it has no absorption at wavelengths above 350 nm[58], [59]In this study, we have reported the synthesis of hydroxylated and carboxylated unsaturated PHAs obtained from the mixtures of 10-undecenoic acid (U) and octanoic acid (OA), U and soybean oil (Sy), and O and Sy by using the photolytic thiol-ene click reaction in the presence of benzophenone. The obtained PHA derivatives with hydroxyl and carboxyl pendant functionalities were characterized by1H-,13C-, HMBC and HSQC NMR, Gel Permeation Chromatog-raphy (GPC) and thermal analysis techniques.

2. Experimental 2.1. Materials

3-Thio glycerol (3TG), mercapto propionic acid (MPA) and the chemicals used in this work were supplied from SigmaeAldrich and used without further puriﬁcation. Soybean oil (Sy) is a com-mercial product obtained from soybean grown in Western Turkey. Soybean oil was hydrolysed in a 10% solution of KOH in ethanol, after which the solution was neutralised with a 10% solution of sulphuric acid in water to obtain the carboxylic acid substrates consisting of oleic acid (25 wt%), linoleic acid (51 wt%) and linolenic acid (9 wt%). Bacterial polyesters were produced by feeding Pseu-domonas oleovorans from 10-undecenoic acid (PHU), the equimolar mixture of soybean oily acids and octanoic acid (PHOSy), the equimolar mixture of soybean oily acids and 10-undecenoic acid (PHUSy), and the equimolar mixture of octanoic acid and 10-undecenoic acid (PHOU) (60). They were all supplied from the TUBITAK-MAM Food Research Institute, Gebze-Kocaeli Turkey. Calculated unsaturation from their1H NMR Spectra:

PHOSy-5050, Mn: 60,000 g/mol, MWD: 2,66. Unsaturation, mol %: 0.8.

PHOU-5050, Mn: 64,000 g/mol, MWD: 3.11. Unsaturation, mol%: 11

PHU-Sy, Mn: 63,000 g/mol, MWD: 2.80. Unsaturation, mol%: 9 PHU, 36,000 g/mol, MWD: 1.92. Unsaturation, mol%: 18

2.2. Polymer characterization

Proton and carbon NMR spectra were acquired at a tempera-ture of 25C with a Agilent NMR 600 MHz NMR (Agilent, Santa Clara, CA, USA) spectrometer equipped with a 3 mm broadband probe. Acquisition parameters included a 45hard pulse angle, a sweep width of 14 ppm, 1.7 s acquisition time, 0.1 s pulse delay and continuous WALTZ-16 broadband 1H decoupling. Up to 2000

Scheme 1. Units of the unsaturated PHA-copolymers PHU (PHOU), PHO-co-PHA-Sy (PHOSy), PHU-co-PHO-co-PHA-Sy (PHUSy). The PHA obtained from soy bean oil con-taining two pendant double bonds was representatively shown in this Scheme. In fact, PHA-Sy contains one, two or three pendant groups related to the fatty acid inclusion of soya oil[29,44,58].

Scheme 2. Schematic design of the carboxylated (I) and hydroxylated (II) PHA derivatives.

Table 1

Diversiﬁcation of the unsaturated PHA-copolymers via thiol-ene click reactions. (Solvent: CH2Cl2; irradiation at 254 nm for 5 h at room temperature. Code PHOU -5050 (g) PHOSy -5050 (g) PHU -100 (g) BzPh (g) MPA (g) 3-TG (g) Yield (g) GPC results

Mn Mw MWD PHOUeOH-1 0.20 e e 0.020 e 0.64 0.10 41,756 136,785 3.275 PHOSy55-COOH-1 e 0.51 e 0.020 1.01 e 0.45 52,052 172,624 3.254 PHU-COOH-1 e e 0.657 0.037 1.57 e 0.62 74,425 441,057 5.926 PHU-OH-1 e e 0.566 0.051 e 1.91 0.58 70,428 292,548 4.154 PHU-OH-2 e e 0.528 0.035 e 1.09 0.69 62,537 214,079 4.423 PHOUeCOOH-1 0.20 e e 0.020 0.68 e 0.13 17,689 182,738 10.33 PHOUeCOOH-2 0.158 e e 0.010 0.35 e 0.23 38,878 132,048 3.396 PHOSy55-OH-1 e 0.50 e 0.020 e 1.02 0.40 65,687 240,335 3.659 PHUSyeCOOHe1 e 0.51 e 0.020 1.01 e 0.377 52,333 175,824 3.360 PHUSyeOHe1 e 0.50 e 0.020 e 1.02 0.317 53,642 324,379 6.047

(3)

scans were collected per sample, corresponding to ~1 h of collection time.

2.3. Gel permeation chromatography (GPC)

Molecular weights were determined by gel permeation chro-matography instrument, Viscotek GPCmax Auto sampler system, consisting of a pump, three ViscoGEL GPC columns (G2000H HR, G3000H HR and G4000H HR), and a Viscotek differential refractive index (RI) detector with a THFﬂow rate of 1.0 mL/min at 30_{C. The} RI detector was calibrated with PS standards having narrow mo-lecular weight distribution. Data were analysed using Viscotek OmniSEC Omnie01 software.

2.4. Thermal analysis

Thermal analysis of the obtained polymers was carried out un-der nitrogen using a TAQ2000 DSC and Q600 Simultaneous DSC-TGA (SDT) series thermal analysis systems. Differential Scanning Calorimeters (DSC) measures temperatures and heatﬂows associ-ated with thermal transitions in the polymer samples obtained. The dried sample was heated from60 to 120_{C under nitrogen} at-mosphere heating from 20 to 600C at a rate of 10C/min. Thermo Gravimetric Analysis (TGA) measures weight loss under nitrogen atmosphere at a rate of 10C/min.

Raman Spectrophotometer, Laser Raman spectra of the solid polymer samples were measured on a Renishaw Invia Raman Spectrophotometer.

2.5. Thiol-ene photo click reactions

Carboxylation and hydroxylation of the unsaturated microbial polyesters (PHU, PHOU and PHUSy) were performed via thiol-ene photo click reactions. As an example for the carboxylation, 0.50 g of PHOU, 1.01 g of mercapto propionic acid (MPA) and 0.020 g of benzophenone (BzPh) were dissolved in 10 mL of CH2Cl2in a pyrex tube. Argon wasﬂushed into the solution for 1 min. The reaction tube was illuminated by using OSRAM PURITEC HNS Germicidal low mercury UV lamp (dominant wavelength 254 nm) at 5 cm distance for 3 h. During the irradiation, the solution was stirred continuously. The reaction tube content was precipitated from methanol, dried under vacuum at room temperature for 24 h. The same reaction procedure was repeated by using 3-thio glycerol (3-TG) in order to obtain hydroxylated PHAs.

3. Results and discussion

The derivatives of the unsaturated medium chain length PHA-copolymers containing pendant hydroxyl and carboxylic acid groups were successfully obtained from PHU, PHOSy, PHUSy, and PHOU (Scheme 1).

Methylene chloride solution of mercapto propionic acid or 3-thio glycerol in the presence of benzophenone were immersed into a pyrex tube and it was irradiated by a mercury lamp in order to prepare PHA derivatives. Schematic representation of the thiol-ene click reaction has been shown inScheme 2.

While benzophenone cannot be used as a photoinitiator in visible light because it has no absorption at wavelengths above 350 nm, Pyrex tubes absorbs the tail of UV lights from 280 nm in order to initiate the photoclick reaction[59,60].Table 1reports the results and conditions of the thiol-ene click reactions.

Carboxyl and hydroxyl derivatives of the unsaturated PHAs were successfully attained in high yield and with high molecular weight in the range from 41,000 to 74,000 as the same as those of the precursors. This shows that the functionalization technique can be

Fig. 1. GPC curves of the PHA derivatives: (a) PHOSyeOHe1, (b) PHOUeOH-1, (c) PHUSyeOHe1, (d) PHU-COOH-1, (e) PHUSyeCOOHe1, (f) PHOSyeCOOHe1, (g) PHOUeCOOH-1, (h) PHOUeCOOH-2, (i) PHU-OH-1.

(4)

Fig. 2.1_{H NMR spectra of (a) PHOU and PHOU derivatives: (b) PHOUeOH-1 and (c) PHOUeCOOH-1.}

(5)

simply performed without the polyester degradation. There is a good agreement with recent results given in the cited work[61]. In addition to this, the GPC curves were mostly unimodal except that of PHU-OH-1 as shown inFig. 1.

InTable 1, PHOUeCOOH-1 had the lower Mn and higher poly dispersity than the others. In order to understand this result, this reaction was repeated with 2 times excess of the mercaptopro-pionic acid under the same conditions (PHOUeCOOH-2 inTable 1). The results obtained was similar to the others. In this case, it can be said that three to four times of excessive mercaptopropionic acid can cause the decrease in Mn with the high polydispersity.

NMR technique was used the structural characterization of the PHA derivatives.1H NMR spectra of the PHA derivatives showed the characteristic signals of eCH2eOH at 3.5e4.0 ppm, eCH2eSe at 2.9 ppm and the eCOOH at 9.8 ppm as shown inFig. 2. Signals of the double bonds at 4.8 and 5.7 ppm dramatically decrease after the modiﬁcation reactions, which is in good agreement with the saturation of double bonds of the PHA by thiol groups.

The characteristic signals of the unsaturation were nearly dis-appeared in the1H NMR spectra after the click reaction. This result can give us an opportunity to guess the mol ratio of the hydroxyl and carboxyl functionality as the same as those of the double bond mol ratio of the precursors: hydroxyl and carboxyl moities in PHOSyeOHe1/COOH-1, PHOUeOH-1/COOH-1, PHU-SyeOHe1/ COOH-1 and PHU-OH-1/COOH-1 are roughly mol%: 0.8, mol%: 11, mol%: 9 and mol%: 18, respectively.

Two dimensional NMR has explained the chemical structure of the hydroxylated and the carboxylated PHAs in detail (Fig. 3). The characteristic signals of eCH2eOH at 3.5e4.0 ppm in 1H NMR spectrum match the signals at 67e71 ppm in13C NMR spectrum. Fig. 4shows the HMBC NMR spectrum of the PHU-COOH-1 sample. We can easily differentiate the ester carbonyl at 176 ppm and car-boxylic acid carbonyl at 197 ppm. Ester carbonyl and free carcar-boxylic acid signals inFigureeSIe8(13C NMR spectrum of PHU-COOH-1) have been seen at 175.994 ppm and 196.814, respectively.

Raman spectra of the polymer samples showed the exact conﬁrmation of the saturated double bonds by the thiol-ene addition reactions (Figs. 5 and 6). The signals at 1642 cm-1 be-longs to the pendant double bonds while the signals at 1660 cm-1 belong to pendant double bonds of unsaturation of the soybean oil. These signals of the double bonds disappeared in case of hydroxyl and carboxyl derivatives of the PHAs.

Thermal properties of the PHA-derivatives were analyzed by using DSC and TGA. DSC analysis of the PHAs is very well known with their glass transition and melting temperature values. Me-dium chain length PHAs show glass transitions in the range between50 and 30_{C. PHA-Soya has Tg at around}₅₀_{C while}

Fig. 4. Two dimensional NMR spectrum of the carboxylated PHU (PHUeCOOH-1).

Fig. 5. Raman spectra of the polymer samples: (a) 100, (b) OH-1, (c) PHU-OH-2, (d) PHU-COOH-1, (e) PHU-Sy-5050, (f) PHU-SyeOHe1, (g) PHU-SyeCOOHe1.

(6)

PHO has Tg at around30_{C. As for Tm values of the copolymers,} PHASy blocks lowered the Tm values to 40C from 58C. When carboxyl and hydroxyl functionalization were performed, the modiﬁed PHAs still exhibited some crystallization. DSC curves of the PHA derivatives are shown inFig. 7. Tg's of the PHA derivatives have varied between from 25 to 38_{C. PHOUeOH-1 sample} exhibited three different Tm values: 45, 58 and 98C. The highest Tm value can come from the hydroxyl functionalization. While carboxyl functionalized PHU has only Tg, hydroxyl functionalized PHU exhibited a higher Tm coming at 58C. Hydroxyl and carboxyl

derivatives of the PHOSy samples have both Tg and Tm at37 and 48C, respectively.

PHOU, PHU and their copolymers exhibit the decompositions in range between 308 and 311C as we reported in a recent article [61]. Hydroxyl and carboxyl derivatives of the PHAs decomposed at the higher temperatures than the precursors as expected. Decomposition temperatures of the PHA derivatives are listed in Table 2.

The hydrophilic character of the modified PHAs was deter-mined by the water drops on the polymerfilms.Fig. 8shows the photographs of the water drops on the modi_{fied PHA films. Less} than 90contact angles for the modified PHAs indicated the in-crease in hydrophilicity, while hydrophobic PHU have at around 90ocontact angles. In this manner, the most dramatic decrease in contact angle was observed in hydroxylated PHUs (PHUeOH-1 and PHU-OH-2).

4. Conclusion

The enhanced hydrophilicity of the PHAs is important for the medical and physicochemical applications. Improved mechanical and hydrophilic character of the unsaturated microbial polyesters can be performed by using thiol-ene photo click reaction. Generally,

Fig. 6. Raman spectra of the polymer samples: (a) PHO, (b) Sy-5050, (c) PHO-SyeCOOHe1, (d) PHO-SyeOHe1, (e) PHOU, (f) PHOUeCOOH-1, (g) PHOUeOH-1.

Fig. 7. DSC thermograms of the PHA derivatives: (a) PHOUeCOOH-1, (b) PHOUeOH-1, (c) PHU-COOH-1, (d) PHU-OH-1, (e) PHOSyeOHe1, (f) PHOSyeCOOHe1.

Table 2

Decomposition temperatures of the PHA derivatives under nitrogen.

PHA-derivative Td1 Td2 PHOUeCOOH-1 314 PHOUeOH-1 324 PHU-COOH-1 319 PHU-OH-1 301 324 OSy55-OH-1 323 OSy55-COOH-1 311

(7)

the hydroxylation and carboxylation modification reactions of the double bonds of the unsaturated PHAs usually lead to dramatic molecular weight decrease. In the present work, we have achieved the synthesis of the hydroxylated and carboxylated PHAs with the same molecular weight of the starting PHAs without chain scission. From the point of this view, thiol-ene photo click modification re-actions are very simple and useful for energy conservation. Pendent hydroxyl and carboxyl groups are also open for further modi fica-tion reacfica-tions in order to prepare novel modified biodegradable polymers for drug delivery system and industrial applications. Acknowledgements

This work was supported by; both the Bülent Ecevit University Research Fund (#BEU-2012-10-03-13) and TUBITAK (Grant # 211T016). The Authors thank to Mahmut K€ose and _Ibrahim Demi-rtas¸ for their valuable discussion; Elvan Akyol for GPC measurements.

Appendix A. Supplementary data

Supplementary data related to this article can be found athttp:// dx.doi.org/10.1016/j.polymdegradstab.2015.04.024.

References

[1] Chen G-Q. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem Soc Rev 2009;38:2434e46.

[2] Gross RA, Kalra B. Biodegradable polymers for the environment. Science 2002;297:803e7.

[3] Langer R, Tirrell DA. Designing materials for biology and medicine. Nature 2004;428:487e92.

[4] Chiellini E, Solaro R. Biodegradable polymeric materials. Adv Mater 1996;8: 305e13.

[5] Solaiman DKY, Ashby RD, Foglia TA, Marmer WN. Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates). Appl Microbiol Bio-technol 2006;71:783e9.

[6] Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B. Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 1988;54:2924e32.

[7] Nguyen S. Graft copolymers containing poly(3-hydroxyalkanoates) d A re-view on their synthesis, properties, and applications. Can Chem-Revue Can Chim 2008;86:570e8.

[8] Koçer H, Borcaklı M, Demirel S, Hazer B. Production of bacterial polyesters from some various new substrates by Alcaligenes eutrophus and Pseudomonas oleovorans. Turk J Chem 2003;27:365e73.

[9] Zinn M, Witholt B, Egli T. Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 2001;53(1):5e21. [10] Wu Q, Wang Y, Chen G-Q. Medical application of microbial biopolyesters

polyhydroxyalkanoates. Artif Cells, Blood Substitutes Biotechnol 2009;37: 1e12.

[11] Steinbüchel A. Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Macromol Biosci 2001;1:1e24. [12] Jossek R, Reichelt R, Steinbüchel A. In vitro biosynthesis of

poly(3-hydroxybutyric acid) by using puri®ed poly(hydroxyalkanoic acid) syn-thase of chromatium vinosum. Appl Microbiol Biotechnol 1998;49: 258e66.

[13] Kim DY, Kim WK, Chung MG, Rhee YH. Biosynthesis, modiﬁcation, and biodegradation of bacterial medium-chain-length polyhydroxyalkanoates. J Microbiol 2007;45:87e97.

[14] Sudesh K, Abe H, Doi Y. Synthesis, structure and properties of poly-hydroxyalkanoates: biological polyesters. Prog Polym Sci 2000;25:1503e55. Fig. 8. Photographs of the water drops on the modiﬁed PHAs.

(8)

[15] Doi Y, Kitamura S, Abe H. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 1995;28: 4822e8.

[16] Lenz RW, Marchessault RH. Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology. Biomacromolecules 2005;6:1e8.

[17] Hazer DB, Kilicay E, Hazer B. Poly (3-hydroxyalkanoate)s: diversiﬁcation and biomedical applications. A state of the art review. Mater Sci Eng C 2012;32: 637e47.

[18] Hazer B, Steinbüchel A. Increased diversiﬁcation of polyhydroxyalkanoates by modiﬁcation reactions for industrial and medical applications. Appl Microbiol Biotechnol 2007;74:1e12.

[19] Hazer B. Poly (b-hydroxy nonanoate) and polystyrene or poly (methyl methacrylate) graft copolymers : microstructure characteristics and me-chanical and thermal behavior. Macromol Chem Phys 1996;197:431e41. [20] Hazer B. Amphiphilic poly (3-Hydroxy Alkanoate)s: potential candidates for

medical applications. Int J Polym Sci 2010. Article Number:423460, doi: 10. 1155/2010/423460.

[21] Nguyen S, Marchessault RH. Synthesis and properties of graft copolymers based on poly(3-hydroxybutyrate) macromonomers. Macromol Biosci 2004;4:262e8.

[22] Nguyen S, Marchessault RH. Atom transfer radical copolymerization of bac-terial poly(3-hydroxybutyrate) macromonomers and methyl methacrylate. Macromolecules 2005;38:290e6.

[23] Arslan H, Adamus G, Hazer B, Kowalczuk M. Electrospray Ionization tandem mass spectrometry of poly [(R,S-hydroxy butanoic acid] telechelics containing primary hydroxyl end groups. Rapid Commun Mass Spectrom 1999;13: 2433e8.

[24] Scandola M, Focarete ML, Adamus G, Sikorska W, Baranowska I, Swierczek S, et al. Polymer blends of natural poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and a synthetic atactic poly (3-hydroxybutyrate). Charac-terization and biodegradation studies. Macromolecules 1997;30:2568e74. [25] Zinn M, Witholt B, Egli T. Occurrence, synthesis and medical application of

bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 2001;53:5e21. [26] Arkin AH, Hazer B. Chemical modiﬁcation of chlorinated microbial polyesters.

Biomacromolecules 2002;3:1327e35.

[27] Li J, Li X, Ni X, Leong KW. Synthesis and characterization of new biodegradable amphiphilic Poly(ethylene oxide)-b-poly[(R)-3-hydroxy butyrate]-b-poly(-ethylene oxide) triblock Copolymers. Macromolecules 2003;36:2661e7. [28] Kim YB, Rhee YH, Lenz RW. Poly(3-hydroxyalkanoate)s produced by

Pseu-domonas oleovorans grown by feeding nonanoic and 10-undecenoic acids in sequence. Polym J 1997;29:894e8.

[29] Hazer B, Torul O, Borcakli M, Lenz RW, Fuller RC, Goodwin SD. Bacterial production of polyesters from free fatty acids obtained from natural oils by Pseudomonas oleovorans. J Environ Polym Degrad 1998;6:109e13.

[30] Kim DY, Kim YB, Rhee YH. Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseu-domonas putida. Int J Biol Macromol 2000;28:23e9.

[31] Fritzsche K, Lenz RW. Productıon of unsaturated polyesters by pseudomonas-oleovorans. Int J Biol Macromol 1990;12:85e91.

[32] Bear M-M, Leboucher-Durand M-A, Langlois V, Lenz RW, Goodwin S, Guerin P. Bacterial poly-3-hydroxyalkenoates with epoxy groups in the side chains. React Funct Polym 1997;34:65e77.

[33] Park WH, Lenz RW, Goodwin S. Epoxidation of bacterial polyesters with un-saturated side chains. I. Production and epoxidation of polyesters from 10-undecenoic acid. Macromolecules 1998;31:1480e6.

[34] Lee MY, Park WH. Preparation of bacterial copolyesters with improved hy-drophilicity by carboxylation. Macromol Chem Phys 2000;201:2771e4. [35] Lee MY, Park WH, Lenz RW. Hydrophilic bacterial polyesters modiﬁed with

pendant hydroxyl groups. Polymer 2000;41:1703e9.

[36] Stigers DJ, Tew GN. Poly(3-hydroxyalkanoate)s functionalize with carboxylic acid groups in the side chain. Biomacromolecules 2003;4:193e5.

[37] Eroglu MS, Hazer B, Ozturk T, Caykara T. Hydroxylation of pendant vinyl groups of poly (3-hydroxy undec-10-enoate) in high yield. J Appl Polym Sci 2005;97:2132e9.

[38] Sparks J, Scholz C. Biomacromolecules 2008;9:2091e6.

[39] Arkin AH, Hazer B, Borcakli M. Chlorination of poly-3-hydroxy alkanoates containing unsaturated side chains. Macromolecules 2000;33:3219e23. [40] Ilter S, Hazer B, Borcakli M, Atici M. Graft copolymerization of methyl

meth-acrylate onto bacterial polyester containing unsaturated side chains. Macro-mol Chem Phys 2001;202:2281e6.

[41] Cakmakli B, Hazer B, Borcakli M. Polystyreneperoxide and poly(methyl methacrylate) peroxide for grafting on unsaturated bacterial polyesters. Macromol Biosci 2001;1:348e54.

[42] Gagnon KD, Lenz RW, Farris RJ. Fuller RC.Chemical modiﬁcation of bacterial elastomers .1. Peroxide cross-linking. Polymer 1994;35:4358e67.

[43] Gagnon KD, Lenz RW, Farris RJ, Fuller RC. Chemical modiﬁcation of bacterial elastomers. 2. Sulfur vulcanization. Polymer 1994;35:4368e75.

[44] Hazer B, Demirel S_I, Borcakli M, Eroglu MS, Cakmak M, Erman B. Free radical crosslinking of unsaturated bacterial polyester obtained from soybean oily acids. Polym Bull 2001;46:389e94.

[45] Hoyle CE, Bowman CN. ThioleEne click chemistry. Angew Chem e Int Ed 2010;49:1540e73.

[46] Schlaad H, Diehl C, Gress A, Meyer M, Demirel AL, Nur Y, et al. Poly(2-oxazoline)s as smart bioinspired polymers. Macromol Rapid Commun 2010;3:511e25.

[47] Yue J, Li X, Mo G, Wang R, Huang Y, Jing X. Modular functionalization of amphiphilic block copolymers via radical-mediated thiol-ene reaction. Mac-romolecules 2010;43:9645e54.

[48] Boyd DA, Naciri J, Fontana J, Pacardo DB, Shields AR, Verbarg J, et al. Facile fabrication of color tunable ﬁlm and ﬁber nanocomposites via thiol click chemistry. Macromolecules 2014;47:695e704.

[49] Lowe AB. Thiol-ene“Click” reactions and recent applications in polymer and materials synthesis: aﬁrst update. Polym Chem 2014;5:4820e70. [50] Keles¸ E, Hazer B, C€omert FB. Synthesis of antibacterial amphiphilic elastomer

based on polystyrene-block-polyisoprene-block-polystyrene via thiol-ene addition. Mater Sci Eng C 2013;33:1061e6.

[51] Campos LM, Killops KL, Sakai R, Paulusse JMJ, Damiron D, Drockenmuller E, et al. Development of Thermal and photochemical strategies for ThiolEne click polymer functionalization. Macromolecules 2008;41:7063e70. [52] Unverferth M, Meier MAR. Tuning the polarity of ADMET derived star-shaped

polymers via thiol-ene Chemistry. Polymer 2014;55:5571e5.

[53] Babinot J, Guigner J-M, Renard E, Langlois V. Poly(3-hydroxyalkanoate)-derived amphiphilic graft copolymers for the design of polymersomes. Chem Commun 2012;48:5364e6.

[54] Babinot J, Renard E, Le Droumaguet B, Guigner J-M, Mura S, Nicolas J, et al. Facile synthesis of multicompartment micelles based on biocompatible poly(3-hydroxyalkanoate). Macromol Rapid Commun 2013;34:362e8. [55] Hazer DB, Hazer B. The effect of gold clusters on the autoxidation of poly

(3-hydroxy10-undecenoate-co-3-hydroxy octanoate) and tissue response eval-uation. J Polym Res 2011;18:251e62.

[56] Wu Q, Wang Y, Chen G-Q. Medical application of microbial biopolyesters polyhydroxyalkanoates. Artif Cells, Blood Substitutes Biotechnol 2009;37: 1e12.

[57] Hazer DB, Hazer B, Dinçer N. Soft tissue response to the presence of polypropylene-g-poly(ethylene glycol) comb-type graft copolymers contain-ing gold nanoparticles. J Biomed Biotechnol 2011;2011:7.http://dx.doi.org/ 10.1155/2011/956169. Article ID 956169.

[58] Hazer B, Hazer DB, Çoban B. Synthesis of microbial elastomers based on soybean oil. Autoxidation kinetics, thermal and mechanical properties. J Polym Res 2010;17:567e77.

[59] Yagci Y, Jockusch S, Turro NJ. Photoinitiated polymerization: advances, chal-lenges, and opportunities. Macromolecules 2010;43:6245e60.

[60] Shih H, Lin C-C. Visible-light-mediated thiol-ene Hydrogelation using eosin-Y as the only photoinitiator. Macromol Rapid Commun 2013;34:269e73. [61] Hany R, Bohlen C, Geiger T, Hartmann R, Kawada J, Schmid M, et al. Chemical

synthesis of crystalline comb polymers from oleﬁnic medium-chain-length poly[3-hydroxyalkanoates]. Macromolecules 2004;37:385e9.