Chemical Modification of Chlorinated Microbial Polyesters

Biomacromolecules 2002, 3, 1327-1335

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Chemical Modification of Chlorinated Microbial Polyesters Ali Hakan Arkin and Baki Hazer* Department of Chemistry, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey Received July 1, 2002; Revised Manuscript Received July 10, 2002

Chlorination of microbial polyesters poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyoctanoate) (PHO) was carried out by passing chlorine gas through their solutions. The chlorine contents in chlorinated PHB (PHB-Cl) and chlorinated PHO (PHO-Cl) were between 5.45 and 23.81 wt % and 28.09 and 39.09 wt %, respectively. Molecular weights of the chlorinated samples were in the range of between one-half to onefourth of the original values because of hydrolysis during the chlorination process. Thermal properties of the PHO-Cl were dramatically changed with an increase in its glass transition (Tg ) 2 °C) and the melting transition (Tm). The Tg of PHB-Cl varied from -20 to 10 °C, and its Tm decreased to 148 °C. The chlorinated poly(3-hydroxyalkanoate)s (PHA-Cl) were converted to their corresponding quaternary ammonium salts (PHA-N+R3), sodium sulfate salts (PHA-S), and phenyl derivatives (PHA-Ph). Cross-linked polymers were also formed by a Friedel-Crafts reaction between benzene and PHA-Cl. The modified PHO derivatives were characterized by 1H NMR and 13C NMR spectrometry, Fourier transform infrared spectroscopy, gel permeation chromatography, and differential scanning calorimetry techniques. Introduction Poly(3-hydroxyalkanoate)s (PHAs) are natural aliphatic polyesters distributed in biological systems and are produced by a wide range of microorganisms as intracellular carbon and energy sources.1-3 These thermoplastic polymers have attracted much attention due to their biodegradability and biocompatibility.1 The general structure of the repeating unit of PHAs is shown below, in which n depends on the substrates and the type of the bacteria.4-9 As the length of the side chain on β-carbon increases, the physical and mechanical properties of PHAs vary from crystalline and brittle to soft and sticky.

synthesized by enzyme-mediated polycondensation,18-20 chlorination,21 epoxidation,22 hydroxylation,23 cross-linking24 of unsaturated side chains, and PHB macromer25 synthesis and ring-opening polymerization.26-30 In our laboratory, medium chain length (mcl) PHAs containing unsaturated side chains have been chlorinated to obtain new modified polyesters.21 In this work, chlorination reactions of short chain length (scl) PHA (PHB) and mcl PHA (PHO) were carried out to obtain PHB-Cl and PHOCl, from which phenyl, quaternary ammonium, and thiosulfate moieties of PHB and PHO were synthesized. Experimental Section

The most well-known PHA is poly(3-hydroxybutyrate) (PHB) in which n ) 0 and which has very high crystallinity (more than 50%) causing low solubility and processing problems.1-3 Poly(3-hydroxyoctanoate) (PHO) in which n ) 4 has low melting transition. A large amount of study has been devoted toward incorporating various functional groups in PHAs. The majority of such modified PHAs were obtained by biofermentation processes using carbon sources bearing corresponding functional groups such as alkene,4,5,10,11 phenyl,7,8 alkyne,12 halogen (fluorine,13 chlorine14), cyano,15 phenoxy,16 and thiophenoxy.17 However, functional PHAs were also * Corresponding author: fax, +90 (372) 323 86 93; e-mail, bhazer@ karaelmas.edu.tr.

Materials. All chemicals were purchased from Aldrich. Acetone, dichloromethane (CH2Cl2), chloroform (CHCl3), carbon tetrachloride (CCl4), methanol (MeOH), sodium thiosulfate pentahydrate (Na2S2O3‚5H2O), potassium permanganate (KMnO4), and metallic sodium (Na0) used as received. Aluminum chloride (AlCl3) was dried over P2O5 under reduced pressure. Triethylamine (NEt3) was dried and distilled over KOH pellets, benzene was dried over Na0, and N,N-dimethylformamide (DMF) was dried over CaH2 and distilled under vacuum. (A) General Procedures. (1) PHA Biosynthesis. Pseudomonas oleoVorans (Deutsche Sammlung von Microorganismen und zell kulturen GmbH, DSM # 1045) and Alcaligenes eutrophus (DSM # 428) were grown in 3 L flasks or a 10 L fermenter at 30 °C in E-2 medium, and the resulting polymers were extracted in a conventional manner according to the procedures cited in the literature.4,7,8 Molecular weight (Mn) of PHB and PHO obtained was 9.7 × 104 and 3.9 × 104; molecular weight distribution (MWD) ) 4.6 and 1.7, respectively.

10.1021/bm020079v CCC: $22.00 © 2002 American Chemical Society Published on Web 08/23/2002

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Arkin and Hazer

Table 1. Results and Conditions for the Chlorination Reactions of PHB and PHO reaction no. PHB 211 21K 231 232 25f 24g PHO 221 191

reactants PHB, g

PHO, g

Cl2, g

yield of PHA-Cl, g

1.23

6

1.9

1.17

17

1.4

2.0 2.0

25 45

2.5 2.3

23 25

7.7 6.5

5.86 3.27

fractional precipitation γa

Cl in PHA-Cl

wt %

0.7-1.0 1.0-1.5 30 1.5-2.0 70 1.0-1.5 25 2.5-4.0 75 0.3-0.7 95 MeOH soluble 1.5-2.0 0.4-0.5 91 0.3-0.5 93

mol %b

wt %c

molecular weighte 10-3Mn 97

MWDf 4.6

22 8d 14 26.9 37.1 22.2 35.1

10.2 8.1 19.3 7.0 39 19.3 25.6

thermal analysis Tg, °C Tm, °C

1.4 1.2 2.4 1.2 1.7 1.7 2.0

2

134

10 -20

148

6 2

a γ is the volume ratio of nonsolvent (MeOH) to solvent (CHCl ). b Calculated from 1H NMR spectra. c Determimed by elemental analysis. d Determined 3 by the Volhard method. e Measured by GPC. f Molecular weight distribution. g Kept in refrigirator for 3 weeks after chlorination.

(2) Chlorination of the PHAs. The chlorination procedure we reported previously21 was used. To the KMnO4 crystals placed in a two-necked round-bottomed flask, excess HCl was added dropwise to produce chlorine gas (1 g of chlorine gas needs 0.89 g of KMnO4). Required moles of the produced gas were passed, at a bubble per second, through the concentrated H2SO4, an empty wash bottle, and then a solution of PHA in CHCl3/CCl4 (80/20 v/v) in an ice bath under sunlight. The estimated amount of Cl2 is shown in Table 1. Chlorinated PHA (PHA-Cl) solution was left in a refrigerator overnight. The solvent was evaporated, and the crude polymer was washed with MeOH, dried under vacuum, and then fractionally precipitated. The precipitated polymer fractions were dried under vacuum. (3) Fractional Precipitations of PHA-Cl were carried out according to the procedure cited in the literature.31 Vacuumdried chlorinated biopolyester sample was dissolved in 5 mL of CHCl3. To the stirring solution, MeOH was added dropwise until completion of the first precipitation. After decantation, the upper solvent was followed by addition of MeOH for the second fraction. The same procedure was attempted until no more precipitation. Gamma (γ) values were calculated as the ratio of the total volume of MeOH used for each fraction to the volume of CHCl3. Polymer fractions were dried under vacuum. (4) Determination of Chlorine Content by the Volhard Method. Chlorinated polymer (50 mg) was fusioned with 30 mg of Na0 to transform -Cl to NaCl. To the reaction content was added 2 mL of distilled water, and then the mixture was acidified with dilute HNO3. Afterward analytical content of NaCl was determined by the titration of standard solution of AgNO3 via the Volhard method.32 (5) Quaternization Reactions of PHA-Cl (PHA-NR3). PHA-Cl quaternization reactions of with triethylamine (or triethanol amine) were carried out either in DMF or in CH2Cl2 according to ref 33. Homogeneous polymer solutions were stirred at room temperature for an hour under argon atmosphere. (6) Synthesis of Sulfonated PHA (PHA-S). The modified procedure reported in ref 34 was used. Na2S2O3‚5H2O (500 mg, 2 mmol) dissolved in a minimum amount of distilled water was added to the solution of 500 mg of PHA-Cl in acetone (20 mL). The mixture was refluxed for 3 h and dried

using a rotary evaporator. The resultant mixture was redissolved in CHCl3 and precipitated from MeOH affording PHA-S. (7) Synthesis of Phenyl Derivatives of PHAs (PHA-Ph). PHA-Ph was obtained according to the Friedel-Crafts reaction. The solution of 150 mg of PHA-Cl in dry benzene (15 mL) was added to a suspension of 100 mg of anhydrous AlCl3 in 10 mL of dry benzene under argon atmosphere and refluxed for 4 h. The solution was filtered, precipitated from MeOH, and dried under vacuum at 30 °C. (B) Polymer Characterization. (1) NMR Spectroscopy. 1 H and 13C NMR spectra were recorded in CDCl3 with TMS internal standard using Varian XL 200 and Varian VCR300 NMR for (PHA-Ph). Polymer concentration was 10 mg/ mL for 1H NMR spectroscopy and 100 mg/mL for 13C NMR spectroscopy. Chemical shifts are given in ppm downfield from TMS. (2) FTIR Spectra. FTIR spectra for all samples were recorded on a Perkin-Elmer 177 IR spectrometer. (3) Molecular Weight Measurements. Molecular weights were measured by gel permeation chromatography (GPC) with a Waters model 6000A solvent delivery system with a model 401 refractive index detector and a mode 730 data module and with two Ultrastyragel linear columns in series. THF was used as the eluent at a flow rate of 1.0 mL/min. Sample concentrations of 0.3% (w/v) and injection volumes of 150 µL were used. A calibration curve was generated with six polystyrene standards (molecular masses were 3 × 106, 2.33 × 105, 2.2 × 105, 2150, 580, and 92 g/mol). The correlation coefficient was 0.994. (4) Thermal Characterizations. DSC and thermogravimetric analysis (TGA) was performed with a DuPont 2910 to determine the glass transition temperatures (Tg), the melting transitions (Tm), and decomposition (Td). Samples were heated from -100 to 160 °C in a nitrogen atmosphere at a rate of 10 °C/min. The Tg reported was the onset temperature in the thermogram. (5) Elemetal Analysis. C and H analyses of the modified bacterial polyesters were carried out by using a Carlo Erba 1106 elemental analyzer. Cyclohexanone 2,4-dinitrophenylhydrazone was used as the calibration standard.

Chlorinated Microbial Polyesters

Figure 1.

1H

Biomacromolecules, Vol. 3, No. 6, 2002 1329

NMR spectrum of a PHB-Cl (sample 232, 14 mol % Cl) recorded in CDCl3.

Figure 2. 200 MHz 1H NMR spectrum of a PHB-Cl (sample 24, 37.1 mol % Cl) recorded in CDCl3. X indicates R-proton signals when X ) Cl.

Results and Discussion Synthesis of PHB-Cl and PHO-Cl. Chlorination reactions were carried out in PHB and PHO solutions in a CHCl3/ CCl4 mixture with Cl2 gas. Results and conditions of the chlorination reactions are listed in Table 1. Substitution reactions of the chlorine atom occurred mainly on H-atoms in the saturated hydrocarbon side chains. When chlorine gas was introduced for the longer reaction times, PHB-Cl had higher Cl content (samples 24 and 25 in Table 1). Chlorination reactions resulted in PHB-Cl and PHO-Cl samples with chlorine content of 14-37.1 and 22.2-35.1 mol %, respectively. PHB-Cl samples 211 and 231 have randomly monochlorinated repeating units, while samples 25 and 24

have multichlorinated repeating units. In higher chlorinated samples, R-chlorinated products together with the multichlorinated side chains in both PHB and PHO were formed (see below for NMR analysis results). Most of the chlorinated samples were white solids, but a PHB-Cl sample 25 was a semielastomeric film at room temperature. Presumably, because of hydrolysis during chlorination process, the molecular weights of the polymers decreased. PHA-Cl samples were fractionated by fractional precipitation to obtain chlorinated polymers of higher molecular weight by varying the solvent-nonsolvent ratio, γ, ranges as shown in Table 1. The Mn of PHB-Cl and PHO-Cl samples fractionated in that manner measured from 8.0 to 19.3 and

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Table 2. Chemical Shifts (ppm) and Copolyester Compositions of the PHB-Cl Samples Using 1H and sample 211 unitsa HB

carbon 1 2

1H

13C

2.35-2.65

169.15 40.79

mol %

13C

2.43-2.66

169.15 40.85

mol %

78

HCB

3 4 5

5.24-5.26 1.26

67.62 19.76 168.57

6

2.35-2.65

36.40

67.65 19.82 168.59

2.66-2.73

36.46

5.26-5.37 3.68

69.43 44.97

22

HDCB

7 8 9

5.26-5.35 3.70

69.40 44.89

14

10

HTCB

11 12 13 14 15 16

2Cl-HDCB + 2Cl-HTCB a

1H

13C

168.14 41.89 43.00 5.32 67.66 1.25-1.42 18.18 166.42 36.21 2.84 36.96 5.44 69.61 3.66-3.72 44.62 164.65 34.15 2.94-2.99 34.97 5.58 70.82 6.00 72.69 164.19 40.32 3.46-3.50 41.10 6.15 74.31 95.45 4.25-4.54 53.35-62.00 2.64-2.73

86 5.23-5.26 1.26

NMR Spectra

sample 25b

sample 232 1H

13C

sample 24b 1H

13C

168.31 42.04 42.83 5.37 67.76 1.27-1.41 18.12 168.07 36.03 2.85 36.68 5.48 69.70 3.65-3.75 44.47 165.99 34.05 3.00 34.81 5.61 70.77 6.03 72.50 164.01 39.94 3.48-3.58 41.22 6.16 74.67 95.33 4.15-4.48 54.30-62.00 2.65-2.71

See the text for abbreviation. b Mole fractions could not be determined.

from 19.3 to 25.6 kDa, respectively. The γ values for both samples 211 and 231 were in a range between 1 and 1.5 (Mn 10.2 and 8.1 kDa), while that for sample 25 (Mn 19.3 kDa) was 03-0.7. Sample 25 with higher γ value had twice the Mn of samples 211 and 231. Chlorinated solvents such as CHCl3 and CCl4 are good solvents for bacterial PHB because of its high crystallinity but are very good solvents for PHB-Cl samples. This increase in solubility can be attributed to the decrease in crystallinity together with an expected increase in solubility with increasing chlorine content of the polyester. Mono- or multichlorinated PHB was soluble in common solvents such as acetone, benzene, CH2Cl2, CHCl3, CCl4, dimethyl sulfoxide (DMSO), DMF, ethyl acetate; slightly soluble in tetrahydrofuran (THF); and insoluble in diethyl ether, n-hexane, and petroleum ether (40/60). In addition, methanol was a good solvent for sample 24 having the highest Cl content, but it was a nonsolvent for the other chlorinated samples containing lower Cl content. In that case, the increase in the hydrolysis of the polyesters was observed by lower Mn. Chloroform was previously reported as nonsolvent for stereoregular fractions of β-chloroalkyl derivatives of PHB,18,26 but in this work, the crystallinity of bacterial PHB was disrupted by the chlorination reaction and PHA-Cl samples with lower Mn were formed by hydrolysis leading to the increase in solubility. NMR Characterization of PHB-Cl. Structural analysis of the chlorinated polyester samples was carried out using 1 H and 13C NMR techniques. The random copolyester compositions of PHB-Cl were also calculated from 1H NMR spectra by comparing with relative peak areas of the methine (-OCH-) protons on the polymer backbone. The relative

peak areas of protons on monochlorinated R-carbons and protons on β-carbons were compared in order to calculate the mole fraction. In PHB-Cl samples, repeating units 3-hydroxybutyrate (HB), 4-chloro-3-hydroxybutyrate (HCB), 4,4-dichloro-3-hydroxybutyrate (HDCB), 4,4,4-trichloro-3hydroxybutyrate (HTCB), 2,4,4-trichloro-3-hydroxybutyrate (2Cl-HDCB), and 2,4,4,4-tetrachloro-3-hydroxybutyrate (2ClHTCB) were determined by using NMR techniques. The total chlorine weight percentages of the PHB-Cl random copolymers were given as the sum of chlorine amounts coming from all chlorinated repeating units. However, the total chlorine amount of PHO-Cl varied from 22.2 to 35.1 mol % as the sum of integral ratios of CHCl, CH2Cl, and CHCl2 groups in NMR spectra. It was difficult to determine the exact amount of chlorinated repeating units in PHO-Cl samples containing a long hydrocarbon chain when compared PHB. PHB-Cl sample 232 (14 mol % Cl) was calculated to have 86 mol % of HB units and 14 mol of HCB units while sample 211 (22 mol % of Cl) was calculated to have 78 mol % of HB and 22 mol % of HCB from their 1H NMR spectra. Figure 1 shows a typical 1H NMR spectrum of the sample 232. The 13C NMR spectrum of sample 232 had a typical -CH2Cl signal at 44.97 ppm. Sample 24 had also signals related to HB, HCB, HDCB, and HTCB units in the 1H NMR spectrum in Figure 2. It was difficult to calculate mole ratios of the chlorinated HB units in higher chlorinated PHB samples because of the low resolution of 1H NMR spectrum. The signals at 1.99-2.43 ppm were attributed to the terminal methyl group of nonchlorinated PHB, which was shifted to lower fields due to the highly chlorinated environment. The chemical shifts of the 1H NMR and 13C NMR resonances of samples 232 and 24 are presented in Table 2.



Figure 3. 200 MHz 1H NMR spectra of PHO-Cl samples recorded in CDCl3: (a) sample 221 (22.2 mol % Cl); (b) sample 191 (35.1 mol % Cl).

Figure 4.

13C

NMR (DEPT) spectra of PHO-Cl samples recorded in CDCl3: (a) sample 221 (22.2 mol % Cl); (b) sample 191 (35.1 mol % Cl).

NMR Characterization of PHO-Cl. The 1H NMR and 13 C NMR spectra of the chlorinated PHO samples showed differences depending on the chlorine content. It is easily observed that methylene (-CH2-) and methyl (-CH3) group signals shifted to the lower fields in both 1H NMR and 13C NMR (DEPT) spectra. Spectra of samples 221 and 191 are shown in Figure 3 and Figure 4, respectively. The 1H NMR spectrum of sample 221 (containing 22.2 wt % Cl) exhibits typical terminal methyl (-CH3) signals between 0.87 and 0.92 ppm, such terminal methyl (-CH3) signals are shifted to 0.96-1.15 ppm due to their environment in PHO-Cl samples. Internal methylene (-CH2-) signals appeared between 1.18 and 1.68 ppm. The signals between 1.7 and 1.78 ppm correspond to the methylene (-CH2-) signals neighboring to terminal chlorinated methyl (-CH2Cl) groups.14 The chemical shifts of the internal methylene (-CH2-) group were between 2.07 and 2.25 ppm due to the electron-withdrawing effect of neighboring

chlorine atoms. The R-CH2 protons are cumulated at 2.66 ppm. The signals were between 3.49 and 3.75 ppm for (-CH2Cl) groups. The chlorine-substituted internal methylene (-CHCl-) groups appeared between 4.06 and 4.55 ppm. The two signals at 5.22 and 5.41 ppm represent the protons placed on β-carbons of the main chain of the polymer. The 1H NMR spectrum of sample 191 (containing 35.1 mol % Cl) showed such similarities. There is only a small signal at 1.24 ppm for terminal -CH3 groups affected by chlorine environment. Internal methylene groups (-CH2-) appeared between 1.63 and 1.83 ppm with low intensity. As in the case of the 1H NMR spectrum of sample 221, the chemical shifts observed between 2.19 and 2.47 ppm are the signals of the internal methylene (-CH2-) groups affecting their neighboring groups having chlorine atoms. At 2.77 ppm R-CH2 protons exhibited a sharp signal. (-CH2Cl) signals could be seen between 3.3 and 3.84 ppm. The chlorine-

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Table 3. Chemical Shifts (ppm) in the 1H NMR and

13C

Arkin and Hazer NMR Spectra of PHO and PHO-Cl Samples

PHO carbon

1H

-CH3

0.87-0.92

-CH2- (side chains)

1.15-1.35 1.47-1.64

-CH2-CO2-

2.58

sample 221 13C

13.98 22.51 24.73 31.52 33.74 39.10

-CH2Cl

0.87-0.92 0.96-1.15 1.18-1.68 1.70-1.78 2.07-2.25 2.66

3.49-3.75

-CHCl-

-OCH-CHCl2 -CCl2-CdO

1H

4.06-4.55

5.20

70.85

169.43

substituted internal methylene (-CHCl-) groups are appeared between 4.06 and 4.74 ppm with high intensity. Typically, β-carbon protons exhibited a signal at 5.45 ppm. Polychlorinated sample 191 has a signal corresponding to terminal (-CHCl2) groups at 5.96 ppm. 13C NMR (DEPT) spectra of the chlorinated PHO samples, namely, samples 221 and 191, are shown in Figure 4, and the main signals are tabulated in Table 3. Thermal Analysis of PHB-Cl and PHO-Cl. The glass transition (Tg) and melting transition (Tm) temperatures of the chlorinated samples are also listed in Table 1. DSC thermograms of the chlorinated samples have been shown in Figure 5. Tg and Tm values of the PHB-Cl samples varied from -20 to 10 °C and from 148 °C, respectively, compared to PHB values of Tm of 175 °C and Tg of 0-4 °C. In the case of the PHO-Cl samples, higher Tg values than those of the precursor PHO were observed. For instance, a Tg value of the PHO-Cl (sample 191) increased to 2 °C compared with those of the PHO precursor of -50 °C. Chlorination of the medium chain length polyesters indicated the increase in Tg and Tm as reported previously as in case of chlorination of PHAs with unsaturated side chains. Interestingly, chlorination of the short chain length PHAs (e.g., PHB) indicated the decrease in Tg and Tm. The Tm values of PHB-Cl samples had a very low melting enthalpy except for a sample of PHBCl (sample 25) with a sharp transition peak (Tm ) 148 °C). We can conclude that some extent of the chlorination of the biopolyesters lowered the polymer crystallinity. Thermogravimetric analysis of the chlorinated samples was also carried out, and the same decomposition temperature (Td) with that of untreated PHA, at around 270 °C, was observed. Chemical Modifications of PHO-Cl. Quaternization. Quaternization reactions of PHA-Cl samples were carried out using triethylamine or triethanolamine either in DMF or

5.22, 5.41

sample 191 13C

11.00 20.87 25.47 30.87 31.45 33.67 35.89 39.01 40.81 41.73 44.44 44.92 48.14 54.39 55.60 58.51 59.80 65.15 66.75 68.34 70.47

169.07

1H

13C

1.24

20.85 23.14 25.48

1.63-1.83 2.19-2.47

36.27

2.77

38.99

3.30-3.84

41.40 44.70 48.00

4.06-4.74

58.04 60.83 61.43 63.32 64.29 68.44

5.45 5.96

70.20 72.29 90.10 168.66

Figure 5. DSC thermograms of the chlorinated polyesters (PHB-Cl: samples 211, 25, and 24; PHO-Cl: samples 191 and 221).

CH2Cl2 under argon atmosphere. Triethylamine caused HCl elimination as well as quaternization. According to the preliminary experiments, it has observed that different amine-solvent systems introduced both quaternized amine and unsaturated groups in PHB and PHO. The 1H NMR spectrum of quaternized sample 191 is shown as an example in Figure 6. The spectrum shows typical triethylamine signals between 1.37 and 1.45 ppm as a triplet for -CH3 and between 3.07 and 3.13 ppm as a quartet for N-CH2- groups.



Figure 6. 200 MHz 1H NMR spectrum of PHAN+R3 recorded in CDCl3. Table 4. Results, Conditions, and Solubility Properties of Quaternization, Friedel-Crafts, and Thiosulfate Reactions of PHA-Cl Samples PHA

reagents

solubilityb

solvent

run no.

sample

amount (g)

type

amount

type

volume, mL

temp, °C

yield (g)

MeOH

CHCl3

ether

THF

N1 N2 N3 N4 N5 Ph1a Ph2 S1 S2

191 191 191 221 232 221 232 191 232

0.50 0.15 0.15 0.50 0.20 0.15 0.30 0.45 0.20

N(Et)3 N(Et)3 N(EtOH)3 N(Et)3 N(Et)3 AlCl3 AlCl3 Na2S2O3‚5H2O Na2S2O3‚5H2O

2 mL 0.5 mL 0.5 mL 2 mL 3 mL 0.1 g 0.1 g 0.5 g 0.5 g

DMF CH2Cl2 DMF DMF DMF benzene benzene acetone acetone

10 15 5 10 3 25 40 15 20

22 3 22 22 60 reflux reflux reflux reflux

0.63 0.16 0.18 0.60 0.22 0.23 0.40 0.71 0.27

s s s s s ns ns ns ns

s s s s s s s s s

s s s s s s s s s

s s s s s s s s s

solubilityb

a

starting polyesters

MeOH

CHCl3

ether

THF

PHB PHO PHB-Cl PHO-Cl

ns ns ns ns

s s s s

ns ns ns s

ns s ss ss

30% cross-linked product obtained. b s, soluble; ss, slightly soluble; ns, nonsoluble.

In addition, between 0.85 and 0.88 ppm terminal methyl signals reappeared and the intensity of (-CH2Cl) signals between 3.57 and 3.60 ppm was enhanced. Solubilities of quaternized polyester samples in some common solvents are listed in Table 4. Phenyl Group Substitution. Samples were refluxed in dry benzene with AlCl3. After the fractional separation, the soluble product was isolated from a small amount of crosslinked polymer and dried under vacuum. The 1H NMR spectrum of the PHO-Ph sample in Figure 8b shows characteristic signals, briefly between 0.88 and 0.92 ppm for terminal -CH3. Between 1.24 and 1.86 ppm typical internal -CH2- groups of PHO units appeared. R-CH2 protons are raised at 2.54 ppm. Between 3.53 and 3.67 ppm -CH2Cl- signals and between 3.94 and 3.99 ppm corresponding internal methylene (-CHCl-) groups can be seen. Methine protons were placed at 5.18 ppm, and peaks between 7.2 and 7.3 ppm are observed for corresponding monosubstituted benzene ring. In Figure 8a 1H NMR spectrum of

4-phenyl-3-hydroxybutyrate (PHB-Ph) is given. In addition to the signals coming from sample 232, monosubstituted benzene signals can be seen between 7.18 and 7.28 ppm. Additionally, typical substituted phenyl signals were observed at 126.29, 126.66, 127.99, and 128.35 ppm in the 13C NMR spectrum of PHB-Ph. Sulfonates, PHA-S. Sodium thiosulfate was reacted with PHB-Cl to obtain the sulfonate derivative of bacterial polyester. The FTIR spectrum of PHA-S in Figure 8 illustrates typical sulfonic acid salt absorption near 1165 cm-1 and OdSdO vibration near 1215 cm-1. The peaks that appear at 1640 and 3440 cm-1 belong to water bands36 due to the hydroscopic natures of S-sulfate salts. FTIR absorbances of S-sulfate salt are given as 1175, 1205, 1640, and 3450 cm-1 in the literature.34 The PHA-S was nonsoluble in MeOH, soluble in THF, ether, and chloroform as shown in Table 4. Chain scission was observed during the reaction. Molecular weight and polydispersity of PHA-S (S1 in Table

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Arkin and Hazer

Figure 7. 200 MHz 1H NMR spectra of (a) PHB-Ph and (b) PHO-Ph recorded in CDCl3.

4) were Mn ) 16100 and Mw/Mn ) 1.6, respectively, while Mn of precursor 191 was 25600 Da. FTIR Spectra. Characteristic FTIR spectra of the chlorinated samples show a typical C-Cl stretching band at 720 cm-1. Carbonyl stretching appears at 1740 cm-1. Figure 8 indicates the FTIR spectrum of sample 211 as an illustrative example of PHA-Cl. For PHA-N+R3, the peaks at 1640, 1215, and 1160 cm-1 can be assigned to the quaternary salts (1640, 1218, and 1154 cm-1 were reported in the literature35). Elemental Analysis. C and H elemental analysis of the modified polyesters were carried out in order to support indirectly the Cl results obtained from NMR spectra. C, H elemental analysis results of the modified polyesters are listed in Table 5. After chlorination and the modification reactions, there was a dramatic decrease in the amount of C and H, when compared to those of the precursors (see Table 5 to compare C, H contents of PHB and PHO with those of the modified ones). By taking a repeat unit basically, Cl content of the PHB-Cl can be calculated from C, H analysis as well as 1H NMR spectra.14 The mole percentages of chlorine calculated from NMR spectra were converted into weight percentage as shown below, and results are listed in Table 5: wt. % of Cl in PHB-Cl ) (mol % of HCB) × 35.5 (mol % of HB) × 86 + (mol % of HCB) × 120.5 Figure 8. FTIR spectra of (a) PHO-Cl (sample 221), (b) PHAN+R3, and (c) PHA S.

There was a good agreement between the results of the


Chlorinated Microbial Polyesters Table 5. Elemental Analysis (C, H) Results of the Modified PHAs (wt %) calculated entrya

C

H

PHB PHB-Cl 232 211 25 24 PHO 221 191 PHA-S S1 S2 PHA-N+R3 N1 N2 N5

55.80

7.03

a

67.57

found C

H

Cl

Clb

50.88 48.15 41.00 34.81

6.50 6.14 4.62 4.21

5.45 8.54 17.21 23.81

5.47 8.34

43.61 34.84

5.80 3.57

28.09 39.09

26.78 43.14

3.50 5.71

43.41 39.18 43.01

5.47 4.90 5.60

9.92

See Table 1 and Table 4 for abbreviations. b From 1H NMR.

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Chemical Modification of Chlorinated Microbial Polyesters

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