1. Characterization of BiodegradablePoly(3-hydroxybutyrate-co- butyleneadipate) Copolymers Obtained from Their Homopolymers by Microwave-assisted TransesterificationGiuseppe Impallomenia, Giovanni Marco Carnemollab, Giuseppe Puzzob,Alberto Ballistrerib*, Lucrezia Martinoc, Mariastella Scandolac. a Istitutodi Chimica e Tecnologia dei Polimeri, Consiglio Nazionale delle Ricerche, Via P. Gaifami 18, 95126 Catania, Italy. b Dipartimento di Scienze del Farmaco, Universit di Catania, Viale A. Doria 6, 95125 Catania, Italy. c Dipartimento di Chimica G. Ciamician, Universit di Bologna, Via Selmi 2, 40126 Bologna, Italy

2. IntroductionNot biodegradable plastics Biodegradableplastics 3. Polyhydroxyalkanoates (PHA)R O Poly(3-hydroxyalkanoates)CH C with R= alkyl or functional group O CH2 nPHASCL: short-chain length PHA 3-5 carbon atomsPHAMCL: medium-chain length PHA 6-14 carbon atoms 4. Physical and chemical properties. Average molecular weight ranging between 5104 and1106 Da Enantiomerically pure Biodegradable and biocompatiblePolymer Tg (C) Tm (C) Cristallinity (%) Extension at break (%)P(3HB)15175 50-80 5P(3HB-co-3HV) -114556 50P(3HB-co-4HB) -715043444PP -1517650400 5. PHAs in medicine and pharmaceuticals.Sutures.Bone graft substitutes.Temporary heart valves.Carrier for drug delivery. 6. Role of PHAs in tissue engineering. 7. Poly(alkylene dicarboxylate)sAliphatic polyesters , semi-crystalline polymers with a wide variety ofphysical, mechanical properties and biodegradability.Poly(alkylenedicarboxylate)s, is polymerizedthrough apolycondensation reaction between a diol, HO-R1-OH, and a dicarboxylicacid, HOOC-R2-COOH, forming polyesters like poly(butylene succinate)(PBS) and poly(butylene adipate) (PBA).OOOOO OO OnnPBSPBAAlthough polyesters with relatively high molecular weight have recentlybeen obtained through polycondensation using metal alkoxides ascatalysts high molecular weight is usually difficult to obtain bypolycondensation and synthesized polymers are left with poormechanical properties. 8. Synthesis of new P(HB-co-BA) copolymers via the acidcatalyzed transesterification of PHB and PBA in solution.CHO OO 3O CH CH C + OCH CH CH CHO CCH CH CH CH C2 n 222 2 2222PHBmPBA 1. PTSAH2O, Chloroform, Toluene (reflux) 2. Azeotropic CH O O O 3OCHCH C O CH CH CH CH O C CH CH CH CH C222222222nmP(HB-co-BA) 9. What are the microwavesThe microwaves are not ionizing electromagnetic waves having a wavelengthbetween 1 mm ( = 300 GHz) and 1 dm ( = 3 GHz), they are located in the area ofthe spectrum between the frequencies of the infrared and the radio waves.The frequency of 2.45 ( 0.05) GHz, corresponding in vacuum at a wavelength () of12.2 cm, is that used for applications in the domestic field, scientific, medical, andfor many industrial processes. 10. Design For Efficient Energy: Energy requirements should berecognized for their environmental and economic impacts andshould be minimized. Synthetic methods should be conducted atambient temperature and pressure. Heating mechanisms heat exchange Heating with MicrowaveBenefits: Energy saving Process Efficiency Restrictions on the use of halogenated solvents 11. Table 1: Reaction time, composition, yield, molecular weight,sequence length and randomness of P(HB-co-BA) copolyestersobtained from a 50/50 mol % mixture of PHB and PBA with 5% w/w ofcatalyst. SampleReaction time a (HB)/(BA) b Yield %Mw x 10-3 c Mw/Mn d LHB eLBA f DR g A10.5+250/5083 16.9 1.1928 74 0.05 A2 1+2 53/4770 17.8 1.46 14.2 28.40.10 A3 2+2 49/5165 17.8 1.377.523 0.18 A4 3+2 45/55 5717.6 1.286.111 0.25 a Reaction times (hours) of the two synthetic steps of hydrolysis and azeotropic dehydration. b Molar composition of the resulting copolyesters as measured by 1H-NMR. c Weight average molecular weights as determined by GPC. d Polydispersity index. e Average block length of HB. f Average block length of BA. g Degree of randomness. 12. Table 2: Reaction time, composition, yield, molecular weight,sequence length and randomness of P(HB-co-BA) copolyestersobtained from a 75/25 mol % mixture of PHB and PBA with 5% w/w ofcatalyst. Sample Reaction time a (HB)/(BA) bYield % Mw x 10-3 c Mw/Mn dLHB e LBA f DR g B10.5+266/34 65 11.4 1.35 23370.07 B2 1+2 64/36 60 11.4 1.31 13170.13 B3 2+2 66/34 47 13.1 1.29 13130.15 B4 3+261/3938 15.7 1.21 5.1 5.4 0.38 a Reaction times (hours) of the two synthetic steps of hydrolysis and azeotropic dehydration. b Molar composition of the resulting copolyesters as measured by 1H-NMR. c Weight average molecular weights as determined by GPC. d Polydispersity index. e Average block length of HB. f Average block length of BA. g Degree of randomness. 13. Table 3: Reaction time, composition, yield, molecular weight,sequence length and randomness of P(HB-co-BA) copolyestersobtained from a 50/50 mol % mixture of PHB and PBA with 10% w/wof catalyst.Sample Reaction time a (HB)/(BA) b Yield %Mw x 10-3 cMw/Mn dLHB eLBA f DR gC10.5+2 49/51 5610.61.45 9.117 0.17C2 1+255/45 42 7.71.36 5.6 9.8 0.28C3 2+243/57 35 5.31.71 3.5 7.8 0.41C4 3+254/46 30 4.91.62 2.2 3.0 0.79aReaction times (hours) of the two synthetic steps of hydrolysis and azeotropic dehydration. b Molar composition ofthe resulting copolyesters as measured by 1H-NMR. c Weight average molecular weights as determined by GPC. dPolydispersity index. e Average block length of HB. f Average block length of BA. g Degree of randomness. 14. a a CH O O O 3f,i O CH CH CO CH CH CH CH O CCH CH CH CH C bc 2 d ne 2 f 2 f 2 e 2g h2i 2 i 2 h2 g mehcb8.07.5 7.0 6.56.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0(ppm)Fig. 1: 200 MHz 1H-NMR spectrum in CDCl3 of P(HB-co-39 mol % BA) (Sample B4). 15. i a CH h 3OO O f O CH CH COCH CH CH CH O CCH CH CH CH C bc 2 d n e 2 f 2 f 2 e 2g h 2 i 2 i 2 h2 gm e a b c gd180170160 150 140 130 120 110 100 908070 60 50 40 30 20 10 0(ppm)Fig. 2: 50 MHz 13C-NMR spectrum in CDCL3 of P(HB-co-39 mol % BA) (Sample B4). 16. HB-A-B B-A-B HB-HB-HBB-A-HB A-HB-HB HB-HB-BHB-A-HBA-HB-BB4B3 B2 B1 174.0 173.5 173.0 172.5 172.0 171.5 171.0 170.5 170.0 169.5 169.0 168.5 (ppm)Fig. 3: 50 MHz 13C-NMR spectral expansion of the carbonyl region of the P(HB-co-BA) copolyester(Samples B1-B4). 17. As in all the 13C-NMR spectra the triad signals were only partially resolved, we basedour sequence analysis on dyads intensities. Designating with IA-B, IA-HB, IHB-B, and IHB-HBthe intensity of signals due to A-B, A-HB, HB-B and HB-HB sequences in the 13C-NMRspectra, and considering the copolyesters as terpolymers made up of the threecomonomers HB, B, and A the average block length of BA and HB units (LBA, LHB), anddegree of randomness (DR) may be defined as: LBA = IA-B/IA-HB + 1LHB = IHB-HB/IHB-B + 1DR = 1/LBA + 1/LHB For a random copolymer of 1:1:1 composition, these parameters are expected to assume the values LBA = 3, LHB = 1.5 and DR = 1 18. Differential scanning calorimetry (DSC) Performed on PHB and PBA homopolymers and on selected HB-BAcopolymers, namely those of series B. Each sample was preliminarily heated to 210C to erase previous thermalhistory, then it was analyzed after both controlled cooling at 10C/min andafter quench cooling. All copolymers show melting endotherms except sample B4, why thiscopolymer has the highest degree of randomness and it is reasonable thatin this sample the development of crystallinity is inhibited. Copolymers B1,B2 and B3 show a melting endotherm at temperatures higher than PBA Tm.This implies that the crystal phase concerned is stable in a range where PBAcrystals cannot exist, hence it is likely due to melting of PHB-type crystals.This attribution is plausible for these B series copolymers that are rich in HBunits (61-66 mol %). The melting temperature of such PHB-type crystals is lower than Tm of thecorresponding homopolymer, showing that these microblock copolymersbehave, in this respect, similarly to random copolymers. 19. Differential scanning calorimetry (DSC)Fig. 4: DSC heating curves (20C/min) of the P(HB-co-BA) copolyesters (Samples B1-B4) and of thereference PHB and PBA homopolymers, after controlled cooling at 10 C/min. 20. MALDI-TOF MS I%xyz= (HBx-By-Az)Na+ x-2-2 (x = 2-6)100 x-3-3 (x = 0-6) x-4-4 (x = 0-6) x-5-5 (x = 0-6) x-6-6 (x = 0-3) x-7-7 (x = 0-1) ^ x-2-1 (x = 4-7) x-1-2 (x = 4-11) + x-4-3 (x = 0-7) x-3-2 (x = 2-8) x-2-3 (x = 1-11) + x-5-4 (x = 0-7) x-3-4 (x = 0-9) + x-6-5 (x = 0-5) x-4-5 (x = 0-7) + x-7-6 (x = 0-3) 50 ^ ^ ^ ^0 580 780 980118013801580 Mass (m/z)Fig. 5: MALDI-TOF mass spectrum of P(HB-co-57 mol % BA) (sample C3) in the mass range580-1580 m/z. 21. 2-6-6 3-6-6I%4-6-5 + 8-3-4 2-7-6 + 6-4-51006-5-4 + 10-2-35-5-51-6-73-5-60-7-7 7-4-4 5002-7-6 + 6-4-55-5-5I%6-5-4 + 10-2-3 4-6-5 + 8-3-4 7-4-49-4-3 + 13-1-2 3-5-6100 3-6-610-3-3 9-3-3 2-6-61-6-7 50 0-7-7 01405 142614471468 1489 1510 Mass (m/z)Fig. 10: MALDI-TOF mass spectra of samples P(HB-co-57 mol % BA) (sample C3, upper trace)and P(HB-co-46 mol % BA) C4 (sample C4, lower trace) in the mass range 1405-1510 m/z. 22. Conclusion By microwave assisted transesterificationwere obtained newbiodegradable and biocompatible copolymer from PHB and PBAhomopolymer. The structures of these copolymers is random or microblock depending theon the experimental condition used. The facile and flexible route described here to synthesize P(HB-co-BA)copolyesters, or potentially higher for lesser reaction times and DR, couldallow to obtain new biodegradable materials based on microbial PHB atrelatively lower cost than those obtainable from fermentation processeswhere engineered bacteria are used. Their weight-average molecular weights range from 5,000 to 17,000 Dawith a structure varying from partially crystalline to totally amorphous andwith a single Tg, making them much easily processable than PHB, andsuggesting their potential use as matrices for drug delivery systems.