Acid Catalyzed Transesterification as a Route to Poly(3

laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry. Rossana Alicata , Tony Barbuzzi , Mario Giuffrida , Albe...
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Biomacromolecules 2002, 3, 835-840

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Acid Catalyzed Transesterification as a Route to Poly(3-hydroxybutyrate-co-E-caprolactone) Copolymers from Their Homopolymers Giuseppe Impallomeni,† Mario Giuffrida,† Tony Barbuzzi,‡ Giuseppe Musumarra,‡ and Alberto Ballistreri*,‡ Istituto per la Chimica e la Tecnologia dei Materiali Polimerici, Consiglio Nazionale delle Ricerche, Viale A. Doria 6, 95125 Catania, Italy, and Dipartimento di Scienze Chimiche, Universita` di Catania, Viale A. Doria 6, 95125 Catania, Italy Received February 19, 2002; Revised Manuscript Received April 24, 2002

Copolymers of (R)-3-hydroxybutyric acid (HB) and -caprolactone (CL) with a composition ranging from 28 to 81 mol % of HB were synthesized by transesterification of the corresponding homopolymers in solution in the presence of 4-toluenesulfonic acid. The copolyesters were characterized with regard to their molecular weights, thermal properties, molar compositions, and average block length of repeating units by gel permeation chromatography (GPC), differential scanning calorimetry, 1H NMR, and 13C NMR, respectively. Random and microblock copolymers could be obtained depending on experimental conditions, with weight-average molecular weights of up to 20 000. The glass transition temperature decreased from 2 to -42 °C as the CL content was increased from 0 to 72 mol %. The melting temperature (Tm) of the PCL phase decreased from 70 to 46 °C as the HB content changed from 0 to 47 mol %, while the Tm of the PHB phase decreased from 177 °C to 163 °C as the CL content changed from 0 to 72 mol %. Matrix-assisted laser desorption ionization time-of-flight mass spectra of GPC fractionated samples allowed us to ascertain that copolymers rich in HB units have mostly hydroxyl and carboxyl end groups, while copolymers rich in CL units have mostly tosyl and carboxyl end groups. Introduction Poly(hydroxyalkanoates) (PHAs) are a class of optically active polyesters that occur in a wide range of bacterial microorganisms.1-4 At present more than 100 different monomeric units as constituents of bacterial PHAs have been described.4-6 The main characteristics of PHAs are their biodegradability, biocompatibility, and other physical properties that range from thermoplastic to elastomeric.7,8 The most known of the PHA family is the homopolymer poly(3-hydroxybutyrate) (PHB),1-4 which has attracted industrial attention as an environmentally degradable plastic for a wide range of agriculture, marine, and medical applications.1,7 However, its melting temperature around 180 °C, close to the thermal decomposition temperature, the stiffness, and the brittleness due to its high crystallinity have limited its practical applications.7 To overcome the above drawbacks of PHB, three approaches have been adopted. One approach is the bacterial synthesis of copolyesters containing hydroxyalkanoate units other than 3-hydroxybutyrate. For example, HB copolyesters containing 3-hydroxyvalerate,1,7 or 4-hydroxybutyrate10 can be produced by Alcaligenes eutrophus. Varying the chemical structure of the monomeric * To whom correspondence should be addressed. Telephone: +39-095738-5032. Fax: +39-095-221541. E-mail: [email protected]. † Consiglio Nazionale delle Ricerche. ‡ Universita ` di Catania.

unit and its amount in the copolymer afford copolyesters with a wide range of melting points and crystallinity. The second approach is blending PHB with other polymers or a plasticizer of lower molecular weight. Poly(ethylene oxide),11,12 poly(vinyl alcohol),13 synthetic atactic PHB,14,15 polysaccharides,16 poly(-caprolactone) (PCL),17,18 poly(vinylphenol),19,20 and poly(vinyl acetate)21,22 have been used for this purpose. The third approach is the chemical synthesis of copolymers. Several block copolymers have been used as compatibilizers of immiscible polymer blends to improve the mechanical properties.23-26 For example, a diblock copolymer of isotactic PHB and PCL was studied.27 The diblock copolymer poly[(R,S)-3-hydroxybutyrate-b-6-hydroxyhexanoate) was proposed as a compatibilizer for the immiscible blend of microbial PHB and PCL.28 In the present study we prepared P(HB-co-CL) copolymers by a transesterification reaction conducted in solution between the two homopolymers PHB and PCL. This transesterification procedure represents an easy and low cost way to obtain new biodegradable polymers with a degree of blockness different from those previously reported. 28,29 These copolymers were characterized by 1H and 13C NMR spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry.

10.1021/bm025525t CCC: $22.00 © 2002 American Chemical Society Published on Web 05/22/2002

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Experimental Section PHB (Mw 400 000, Mw/Mn ) 2.1), PCL (Mw 120 000, Mw/ Mn ) 1.7), and 4-toluenesulfonic acid monohydrate were obtained from Aldrich Chemical Co. All other chemicals were of the highest purity commercially available and were used without further purification. Preparation of P(HB-co-CL). Seven grams of PHB/PCL mixtures with molar ratios (in repeating units) of 25/75, 40/ 60, 50/50, 60/40, and 75/25 (samples 1-5) and 5% (w/w) of 4-toluenesulfonic acid monohydrate were dissolved in 150 mL of toluene/dichloroethane (3:1). In a first step the mixtures were stirred at reflux for 4 h, then the water was removed azeotropically with a Dean-Stark trap for 2 h. Sample 6 with molar ratio 50/50 was maintained in the first step for 14 h and for 12 h in the second. The solvent was evaporated, and the crude product was dissolved into a small volume (5 mL) of chloroform. This solution was precipitated in ethanol (50 mL). The product was reprecipitated three times and then dried in vacuo at 50 °C for 24 h. Molecular Weight Measurements. Average molecular weights were determined by GPC using a Waters 515 HPLC pump with 4 Styragel HR columns connected in series (in the order: HR4, HR3, HR2, and HR1) and a model 401 refractive index detector. Chloroform was used as eluent at a flow rate of 1.0 mL/min; 200 µL of a 5 mg/mL solution was injected for each sample. A molecular weight calibration curve was generated with polystyrene standards of low polydispersity (Polymer Laboratories) using the GPC Caliber acquisition and processing software (Polymer Laboratories). Fractionation of samples for subsequent MALDI-TOF MS was performed by collecting 70 fractions of 0.16 mL each. Thermal Analysis. Differential scanning calorimetry was performed with a DSC Du Pont 9900 thermal analyzer equipped with a cooling accessory under a nitrogen flow of 20 mL/min. Samples of 10-15 mg were encapsulated in aluminum pans and heated from 0 to 200 °C at a rate of 10 °C/min (first scan). To determine the glass-transition temperature (Tg), the samples were maintained at 200 °C for 1 min and then rapidly quenched at -80 °C. They were then reheated from - 80 to 200 °C at a heating rate of 20 °C/ min (second scan). The temperature scale was calibrated with high-purity standards. The melting temperature (Tm) was taken at the peak of the melting endotherm, while the Tg was taken as the midpoint of the heat capacity change. Nuclear Magnetic Resonance Spectroscopy. The 200 MHz 1H NMR spectra of the samples were recorded at room temperature in CDCl3 (40 mg/mL) on a Bruker AC200 spectrometer with a 4 s pulse repetition, a 2000 Hz spectral width, 16K data points, and 256 scan accumulation. The 1Hdecoupled 50 MHz 13C NMR spectra were recorded on the same samples with a 1.6 s acquisition time, 10 000 Hz spectral width, 32K data points and 30 000 scan accumulation. MALDI-TOF Mass Spectrometry. The MALDI-TOF mass spectra of the copolyesters were acquired using a Perseptive Voyager-DE-STR instrument equipped with a nitrogen laser emitting at 337 nm with a 3 ns pulse width and working in positive mode. Spectra were recorded in

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linear mode with 128 laser shot accumulation, 25 kV acceleration potential, and laser irradiance slightly above threshold. Spectra were calibrated using narrowly dispersed poly(methyl methacrylate) GPC standards of suitable molar mass. Samples were dissolved in THF/CHCl3, 1:1 (10 mg/ mL), and mixed with the matrix 2-(4-hydroxyphenyl)azobenzoic acid (HABA) (0.1 M in THF/CHCl3, 1:1). A 10 µL portion of the sample solution was added to 30 µL of the matrix solution, and 1 µL of the resulting mixture was deposited on the sample holder and allowed to dry at room temperature. In the case of samples from GPC fractionation, they were prepared as follows: Each collected fraction was evaporated to dryness and then dissolved in 50 µL of THF/ CHCl3 1:1; 10 µL of this solution was treated as described above. Results and Discussion P(HB-co-CL) copolymers with HB content ranging from 28 to 81 mol % were obtained by transesterification between the corresponding homopolymers PHB and PCL in the presence of 4-toluenesulfonic acid monohydrate (TSA). It is known that TSA in dilute solution of PHB catalyzes cyclic oligomer formation.30 We speculated that successful synthesis of copolyesters via transesterification of homopolyesters requires higher concentrations and shorter reaction times. The synthesis we devised occurs in two stages: First, a solution of the two polymers in toluene/dichloroethane 3:1 in the presence of TSA is heated at reflux temperature. Both hydrolysis and transesterification proceed during this stage, as showed by the molecular weight reduction and by the appearance of heterodyad signals in 13C NMR spectra, respectively. In the second stage, water is removed through a Dean-Stark trap, and as a consequence, there is an increase in molecular weight and degree of transesterification (DT, vide infra for definition). When the first stage was omitted, and the reaction was conducted in anhydrous conditions from the beginning, no ester interchange was detected for reaction times up to 24 h. It may be concluded that the reaction occurs via the acid-catalyzed nucleophilic attack of the terminal alcoholic groups on the ester moieties. Without previous hydrolysis the concentration of such groups is too low in order for the transesterification to proceed. The amount of water in the system plays a crucial role. In the first stage it determines the extent of depolymerization of PHB and PCL, which in turn determines the concentration of terminal hydroxyls; in the second stage its removal shifts the equilibrium toward the ester linkage formation. In Table 1 we show the results for six mixtures of homopolymers with various HB/CL mole ratios, reporting the composition and yield of the copolymers obtained, and their weight-average molecular weight (Mw), polydispersity (Mw/Mn), and DT at the end of the two transesterification stages. The HB/CL molar ratios in the isolated copolyesters were nearly identical to that of the starting material. Yields of samples 1-5 varied from 48 to 80%; the loss of material is due to low molecular weight products which do not precipitate in ethanol during the product purification procedure, and its variation may be ascribed to the interplay of various factors such as the

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Table 1. Transesterification Conditions, Yields, Molecular Weights, and Degree of Transesterification of P(HB-co-CL) Copolymers sample

(HB/CL)a

(HB/CL)b

timec

yield, %

Mw × 10-3 d

Mw/Mnd

DTd

1 2 3 4 5 6

25/75 40/60 50/50 60/40 75/25 50/50

28/72 45/55 53/47 68/32 81/19 43/57

4/2 4/2 4/2 4/2 4/2 14/12

68 54 48 61 80 83

14.7 (14.2) 20.1 (16.0) 19.5 (11.8) 13.9 (9.6) 10.6 (8.1) 4.6 (ND)e

1.72 (1.60) 1.55 (1.57) 1.60 (1.52) 1.68 (1.64) 1.63 (1.55) 1.45 (ND)e

0.041 (0.034) 0.114 (0.071) 0.136 (0.087) 0.113 (0.074) 0.079 (0.064) 0.484 (ND)e

a Molar composition of the initial homopolymers mixture. b Molar composition of the resulting copolymers. c Duration in hours of the two transesterification stages. d Weight-average molecular weight, molecular weight distribution, and degree of transesterification at the end of the second stage of the reaction. In parentheses are indicated the corresponding values at the end of the first stage. See eq 2 for definition of DT. e Not determined.

Figure 1. 200 MHz 1H NMR spectrum in CDCl3 of the P(HB-co-47 mol % CL) copolymer (sample 3).

different hydrolysis rate of the two homopolymers, the composition of the mixtures, and a not perfectly controlled amount of water in the system. There was a significant molecular weight reduction with respect to the original PHB and PCL. However, the Mw values of the materials obtained were still high enough for such uses as blend compatibilizers or drug release matrixes. In preliminary experiments (data not reported) we saw that higher Mw could be obtained by reducing the reaction times or the amount of TSA, but at the expense of the degree of transesterification. Figure 1 shows the 1H NMR spectrum of sample 3 with the corresponding assignments. Besides the signals of HB and CL, two doublets at 7.35 and 7.79 ppm sharing the same coupling constant (8.2 Hz) are present. These signals remained unaltered after repeated dissolution in chloroform and reprecipitation in ethyl alcohol and may be assigned to a tosyl group ester linked to the alcoholic moiety of terminal units. The methyl singlet of the TSA ester was found at 2.45 ppm, overlapped with the methylene signal of HB. The presence of tosyl groups chemically linked to the polymer was further demonstrated by the permanence of the proton signals described above in the NMR spectra of fractions isolated by gel permeation chromatography. From the peak areas of the aromatic signals, it was possible to estimate the number average molecular weight (Mn) of samples 1-6, obtaining values consistent with those determined by GPC for the samples rich in CL units but that were increasingly inaccurate in excess for the samples rich in HB units. There was a directly proportional correlation between the area of the tosyl aromatic signals and the CL content in the samples. We hypothesized that the tosyl groups are linked to CL

Figure 2. 50 MHz 13C NMR spectrum in CDCl3 of the P(HB-co-47 mol % CL) copolymer (sample 3).

Figure 3. 13C NMR spectral expansion of the carbonyl region of the P(HB-co-CL) samples 1-6.

terminal units, while terminal HB units have alcoholic terminal groups, though these were not identifiable in the 1 H NMR spectra. Sequence and evidence for transesterification were obtained from 13C NMR. Figure 2 shows the 13C NMR spectrum of sample 3 with the corresponding assignments. The weak signals at 127.8 and 129.8 ppm belong to the aromatic methine carbons of terminal TSA; their chemical shifts are quite similar to those of TSA ethyl ester, taken as a model compound. The methyl resonated at 21.58 ppm. Figure 3 shows an expansion of the carbonyl region for samples 1-6. In the uppermost trace, relative to sample 6, we report the triads assignment according to literature.29 As

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Table 2. Average Block Length and Degree of Randomness of the P(HB-co-CL) Copolymers with Various (HB)/(CL) Molar Ratios sample 1 2 3 4 5 6

(HB)/(CL)a

LBb

LCc

DRd

28/72 45/55 53/47 68/32 81/19 43/57

17.5 9.6 9.1 13.3 21.9 2.0

30.8 8.0 5.6 4.3 3.5 2.1

0.09 0.23 0.29 0.31 0.33 0.97

a Molar composition of the copolymers. b Average block length of HB. See eq 1 for definition. c Average block length of CL. See eq 1 for definition. d Degree of randomness. See eq 3 for definition.

the triad signals were only partially resolved, we based our sequence analysis on dyads intensities. Designating with IBB, IBC, ICB, and ICC the normalized intensities of signals due to HB-HB, HB-CL, CL-HB, and CL-CL sequences, we used the Yamadera and Murano definitions of average block length of HB and CL units (LB and LC), DT, and degree of randomness (DR)31 LB ) 2PB/(IBC + ICB)

(1)

LC ) 2PC/(IBC + ICB) (where PB and PC are the dyad mole fractions of HB and CL, i.e. PB ) IBB + 1/2(IBC + ICB) and PC ) ICC + 1/2(IBC + ICB) DT ) IBC + ICB

(2)

DR ) 1/LB + 1/LC

(3)

For a random copolymer of 1:1 composition, these parameters are expected to assume the values LB ) LC ) 2, DT ) 0.5, and DR ) 1. Table 2 shows the values determined for samples 1-6. From these data it is possible to conclude that when the two stages of the ester exchange reaction are allowed to proceed for longer times, as for sample 6, a random copolymer is obtained, as indicated by the values of DT (Table 1), LB, LC, and DR (Table 2), practically coincident with those theoretically expected. For shorter reaction times and varying monomer ratios (samples 1-5), the DT and DR values indicate a deviation from randomness, and the average length of like monomers in the polymer chain goes up, showing a microblock character for these samples. It looks, therefore, that the reaction may be finely tuned in order to obtain a poly(HB-co-CL) of particular composition or degree of randomness. These copolyesters were thermally characterized by DSC. The first heating thermograms are shown in Figure 4, and the values of the thermal transitions are presented in Table 3. The first heating scans on the samples showed some complexity. The lowest Tm, belonging to the melting of a PCL phase, decreased from 70 (Tm of pure PCL) to 46 °C with increasing amount of HB, while the highest Tm, belonging to a PHB phase, decreased from 177 (Tm of pure

Figure 4. DSC thermograms of the first heating scan of the P(HBco-CL) copolymers of different molar composition (samples 1-5).

PHB) to 163 °C as the amount of CL increased. At intermediate temperatures a bimodal endotherm was found, which may be assigned to the melting of copolymer phases with varying degrees of composition and microblock size. In the second heating on melt-quenched samples, only one glass transition was observed at a temperature between those of the pure homopolymers (for PCL Tg ) -70 °C, for PHB Tg ) 2 °C). This Tg changed with composition and is due to a single amorphous phase resulting from the mixing of various phases during the first heating. Finally, we used MALDI-TOF mass spectrometry to determine the end groups present in these samples. MALDI, introduced in 1988,32,33 is capable of putting in the gas phase polymeric materials which are polydisperse with regard to molecular weight. However, a limit is very often reached with such samples, where the measured molecular weight is grossly underestimated, and the quality of the spectra is severely reduced. It has been shown that this difficulty may be overcome by fractionating by GPC the polydisperse material and applying MALDI to the narrowly dispersed fractions.34,35 The MALDI-TOF mass spectra of samples 1-6 were indeed of poor quality, showing a series of peaks decaying in intensity from m/z 500 (where a mass cutoff was applied) to about m/z 2000-2500. We therefore fractionated the samples and recorded the spectra of a few fractions. Figure 5 shows the MALDI-TOF mass spectrum of a GPC fraction of sample 3 eluting near the maximum of the GPC trace. The spectrum shows two ion distributions: the first is centered at m/z 3700 and is due to tosyl-terminated and sodium-cationized molecules: CH3-Ph-SO2-[O-(CH2)5-CO]m-[O-CH(CH3)CH3-CO]n-OH‚Na+ 1

The second is centered at m/z 4600 and is due to alcoholterminated and sodium-cationized molecules: H-[O-CH(CH3)CH2-CO]m-[O-(CH2)5-CO]n-OH‚Na+ 2

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Table 3. Thermal Data Obtained by DSC of the P(HB-co-CL) Copolymers with Different Compositions sample

CL mol %

Tg,a °C

1 2 3 4 5

72 55 47 32 19

-42 -45 -39 -30 -22

Tmb (∆Hm)c 60 (74.1) 51 (17.3) 46 (11.2) ND ND

100 (3.2) 92 (7.9) 90 (12.6) 91 (15.4) 97 (8.3)

125 (9.0) 109(7.1) 111 (16.0) 117 (19.6) 133 (38.8)

163 (0.9) 165 (6.7) 166 (10.7) 168 (16.1) 168 (8.7)

a Glass-transition temperature (second scan) measured from -80 to 200 °C at a heating rate of 20 °C/min. b Melting temperature (°C, first scan) measured from 0 to 200 °C at a heating rate of 10 °C/min. c Enthalpy of fusion (J/g).

Figure 5. Positive ion MALDI-TOF mass spectrum of a GPC fraction of the P(HB-co-47% mol CL) copolymer (sample 3). The mass assignment of the two spectral expansions is given in Table 4.

which is the difference between the molecular weights of the two repeating units. These results confirm the presence of tosyl and alcoholic end groups. The fact that two populations of molecules with different average molecular weight elute together may be explained as an effect of their different end groups: the tosyl moiety is stiffer and bulkier than the -OH group, or there may be a weak interaction of the tosyl group with the styrene/divinylbenzene resin of the GPC columns. MALDI-TOF mass spectra of GPC fractions of other copolymers gave the same results (data not shown), the samples rich in HB units giving mostly hydroxyl- and carboxyl-terminated species, and the samples rich in CL units giving mostly tosyl- and carboxyl-terminated species, strengthening the evidence gathered by NMR analysis, i.e., that the tosyl groups are linked only to terminal CL units.

Table 4. Mass Assignment of the MALDI-TOF Mass Spectrum of a GPC Fraction of P(HB-co-47 Mol % CL) (Sample 3)

Conclusions

m /z

HBa

CLa

3771.4 3773.3 3775.2 3801.3 3803.3 3805.2 3829.4 3831.3 3833.2 3857.4 3859.4 3861.3 3885.5 3887.4 3889.4 3913.6 3915.5 3917.4 3943.5 3945.5 3947.4 3971.6 3973.5 3975.4

19 23 27 22 26 30 21 25 29 20 24 28 19 23 27 18 22 26 21 25 29 20 24 28

17 14 11 15 12 9 16 13 10 17 14 11 18 15 12 19 16 13 17 14 11 18 15 12

m /z

HBb

CLb

4500.3 4502.3 4504.2 4530.3 4532.2 4534.2 4558.4 4560.3 4562.2 4588.4 4590.3 4592.2 4616.4 4618.3 4620.3 4644.5 4646.4 4648.3 4672.5 4674.4 4676.4 4702.5 4704.4 4706.4

16 20 24 19 23 27 18 22 26 21 25 29 20 24 28 19 23 27 18 22 26 21 25 29

27 24 21 25 22 19 26 23 20 24 21 18 25 22 19 26 23 20 27 24 21 25 22 19

a Number of HB and CL units in structure 1 shown in the text. b Number of HB and CL units in structure 2 shown in the text.

Every peak in the spectrum results from the overlapping of pseudomolecular ions which differ of two amu due to different HB/CL composition. Table 4 reports the mass assignments, showing, for each peak of the expanded regions of Figure 5, only the three compositions which contribute mostly. Significantly, the signals are separated by ca. 28 amu,

A new facile and flexible route to P(HB-co-CL) copolymers has been described starting from PHB and PCL. The new synthesis is based on the acid-catalyzed transesterification of the two homopolymers leading to random or microblock copolymers, depending on the experimental conditions used. Despite the copious scientific literature and industrial enterprise, the impact on the market of PHAs has been so far limited, if not negligible, mainly due to their cost. The copolyesters described here could be used as compatibilizers in blends of PHB with PCL, an inexpensive biodegradable and biocompatible plastic, giving the PHA industry new momentum. Besides this application, these materials are per se interesting, for instance as candidates for drug release matrixes. Studies are being conducted to this aim. Acknowledgment. Partial financial support from the Consiglio Nazionale delle Ricerche (CNR, Rome), the Ministero dell’Istruzione Universita` e Ricerca (MIUR, Rome), and the European Economic Community (contribution no. 94.05.09.013 ARINCO no. 94.IT.16028) is gratefully acknowledged. References and Notes (1) Doi, Y. Microbial Polyesters; VCH Publishers: New York, 1990. (2) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. (3) Brandl, H.; Gross, R. A.; Lenz, R. W.; Fuller, R. C. AdV. Biochem. Eng. Biotechnol. 1990, 41, 77. (4) Steinbu¨chel, A.; Valentin, H. E. FEMS Microbiol. Lett. 1995, 128, 219. (5) Steinbu¨chel, A. Macromol. Biosci. 2001, 1, 1. (6) Madison, L. L.; Huisman, G. W. Microbiol. Mol. Biol. ReV. 1999, 63, 21.

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