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 half to one-fourth 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 Tgof PHB-Cl varied from -20 to 10°C, and its Tmdecreased 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 by1H NMR and13C 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.1-3These thermoplastic polymers have
attracted much attention due to their biodegradability and biocompatibility.1The general structure of the repeating unit
of PHAs is shown below, in which n depends on the substrates and the type of the bacteria.4-9As 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.
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 biofer-mentation processes using carbon sources bearing corre-sponding functional groups such as alkene,4,5,10,11phenyl,7,8
alkyne,12halogen (fluorine,13chlorine14), cyano,15phenoxy,16
and thiophenoxy.17 However, functional PHAs were also
synthesized by enzyme-mediated polycondensation,18-20
chlorination,21epoxidation,22hydroxylation,23cross-linking24
of unsaturated side chains, and PHB macromer25synthesis
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.21In this work, chlorination
reactions of short chain length (scl) PHA (PHB) and mcl PHA (PHO) were carried out to obtain PHB-Cl and PHO-Cl, from which phenyl, quaternary ammonium, and thiosul-fate moieties of PHB and PHO were synthesized.
Experimental Section
Materials. All chemicals were purchased from Aldrich. Acetone, dichloromethane (CH2Cl2), chloroform (CHCl3),
carbon tetrachloride (CCl4), methanol (MeOH), sodium
thiosulfate pentahydrate (Na2S2O3‚5H2O), potassium
per-manganate (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.
Pseudo-monas oleoVorans (Deutsche Sammlung von
Microorgan-ismen und zell kulturen GmbH, DSM # 1045) and
Alcali-genes 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.4,7,8Molecular weight
(Mn) of PHB and PHO obtained was 9.7× 104and 3.9×
104; molecular weight distribution (MWD) ) 4.6 and 1.7,
respectively.
* Corresponding author: fax, +90 (372) 323 86 93; e-mail, bhazer@ karaelmas.edu.tr.
10.1021/bm020079v CCC: $22.00 © 2002 American Chemical Society Published on Web 08/23/2002
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(2) Chlorination of the PHAs. The chlorination procedure we reported previously21was used. To the KMnO
4crystals
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
Vacuum-dried 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 AgNO3via 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 CH2
-Cl2 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 redis-solved 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 AlCl3in 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.
1H and13C NMR spectra were recorded in CDCl
3with TMS
internal standard using Varian XL 200 and Varian VCR-300 NMR for (PHA-Ph). Polymer concentration was 10 mg/ mL for1H NMR spectroscopy and 100 mg/mL for13C 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 150µL 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 thermogravi-metric 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-dinitrophenyl-hydrazone was used as the calibration standard.
Table 1. Results and Conditions for the Chlorination Reactions of PHB and PHO
reactants
fractional
precipitation Cl in PHA-Cl molecular weighte thermal analysis reaction no. PHB, g PHO, g Cl2, g yield of PHA-Cl, g γa wt % mol %b wt %c 10-3M n MWDf Tg,°C Tm,°C PHB 0.7-1.0 97 4.6 211 1.23 6 1.9 1.0-1.5 30 22 2 134 21K 1.5-2.0 70 231 1.17 17 1.4 1.0-1.5 25 8 d 10.2 1.4 232 2.5-4.0 75 14 8.1 1.2 25f 2.0 25 2.5 0.3-0.7 95 26.9 19.3 2.4 10 148 24g 2.0 45 2.3 MeOH soluble 37.1 7.0 1.2 -20 PHO 1.5-2.0 39 1.7 221 5.86 23 7.7 0.4-0.5 91 22.2 19.3 1.7 6 191 3.27 25 6.5 0.3-0.5 93 35.1 25.6 2.0 2
aγis the volume ratio of nonsolvent (MeOH) to solvent (CHCl
3).bCalculated from1H NMR spectra.cDetermimed by elemental analysis.dDetermined
by the Volhard method.eMeasured by GPC.fMolecular weight distribution.gKept in refrigirator for 3 weeks after chlorination.
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). Chlorina-tion reacChlorina-tions resulted in PHB-Cl and PHO-Cl samples with chlorine content of 14-37.1 and 22.2-35.1 mol %, respec-tively. 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 multichlo-rinated 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 precipita-tion to obtain chlorinated polymers of higher molecular weight by varying the solvent-nonsolvent ratio,γ, ranges
as shown in Table 1. The Mnof PHB-Cl and PHO-Cl samples
fractionated in that manner measured from 8.0 to 19.3 and Figure 1. 1H NMR spectrum of a PHB-Cl (sample 232, 14 mol % Cl) recorded in CDCl
3.
Figure 2. 200 MHz1H NMR spectrum of a PHB-Cl (sample 24, 37.1 mol % Cl) recorded in CDCl
from 19.3 to 25.6 kDa, respectively. Theγ values for both
samples 211 and 231 were in a range between 1 and 1.5 (Mn10.2 and 8.1 kDa), while that for sample 25 (Mn19.3
kDa) was 03-0.7. Sample 25 with higherγ value had twice
the Mnof 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 increas-ing chlorine content of the polyester. Mono- or multichlo-rinated PHB was soluble in common solvents such as acetone, benzene, CH2Cl2, CHCl3, CCl4, dimethyl sulfoxide
(DMSO), DMF, ethyl acetate; slightly soluble in tetrahy-drofuran (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 ofβ-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
1H and 13C NMR techniques. The random copolyester
compositions of PHB-Cl were also calculated from1H 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-3-hydroxybutyrate (HTCB), 2,4,4-trichloro-3-4,4,4-trichloro-3-hydroxybutyrate HDCB), and 2,4,4,4-tetrachloro-3-hydroxybutyrate (2Cl-HTCB) were determined by using NMR techniques. The total chlorine weight percentages of the PHB-Cl random copoly-mers 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 typical1H NMR spectrum of the sample
232. The13C 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 the1H 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 of1H 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 the1H NMR and13C NMR resonances of
samples 232 and 24 are presented in Table 2. Table 2. Chemical Shifts (ppm) and Copolyester Compositions of the PHB-Cl Samples Using1H and13C NMR Spectra
sample 211 sample 232 sample 25b sample 24b
unitsa carbon 1H 13C mol % 1H 13C mol % 1H 13C 1H 13C
HB 1 169.15 169.15 168.14 168.31 2 2.35-2.65 40.79 2.43-2.66 40.85 2.64-2.73 41.89 2.65-2.71 42.04 78 86 43.00 42.83 3 5.24-5.26 67.62 5.23-5.26 67.65 5.32 67.66 5.37 67.76 4 1.26 19.76 1.26 19.82 1.25-1.42 18.18 1.27-1.41 18.12 HCB 5 168.57 168.59 166.42 - 168.07 36.21 36.03 6 2.35-2.65 36.40 2.66-2.73 36.46 2.84 2.85 22 14 36.96 36.68 7 5.26-5.35 69.40 5.26-5.37 69.43 5.44 69.61 5.48 69.70 8 3.70 44.89 3.68 44.97 3.66-3.72 44.62 3.65-3.75 44.47 HDCB 9 164.65 165.99 34.15 34.05 10 2.94-2.99 3.00 34.97 34.81 11 5.58 70.82 5.61 70.77 12 6.00 72.69 6.03 72.50 HTCB 13 164.19 164.01 40.32 39.94 14 3.46-3.50 3.48-3.58 41.10 41.22 15 6.15 74.31 6.16 74.67 16 95.45 95.33 2Cl-HDCB+ 2Cl-HTCB 4.25-4.54 53.35-62.00 4.15-4.48 54.30-62.00
aSee the text for abbreviation.bMole fractions could not be determined.
NMR Characterization of PHO-Cl. The1H NMR and 13C 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 both1H NMR and13C
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 environ-ment 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-CH2protons are cumulated at 2.66
ppm. The signals were between 3.49 and 3.75 ppm for (-CH2Cl) groups. The chlorine-substituted internal
meth-ylene (-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-CH2protons exhibited a sharp signal. (-CH2Cl) signals
could be seen between 3.3 and 3.84 ppm. The chlorine-Figure 3. 200 MHz1H 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 CDCl
substituted internal methylene (-CHCl-) groups are ap-peared 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. Tgand Tmvalues of the PHB-Cl samples varied
from -20 to 10°C and from 148°C, respectively, compared to PHB values of Tmof 175°C and Tg of 0-4°C. In the
case of the PHO-Cl samples, higher Tgvalues than those of
the precursor PHO were observed. For instance, a Tgvalue
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 Tgand Tmas reported previously as in case of chlorination
of PHAs with unsaturated side chains. Interestingly, chlo-rination of the short chain length PHAs (e.g., PHB) indicated the decrease in Tgand Tm. The Tmvalues of PHB-Cl samples
had a very low melting enthalpy except for a sample of PHB-Cl (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. Thermo-gravimetric 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
CH2Cl2under 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.
Table 3. Chemical Shifts (ppm) in the1H NMR and13C NMR Spectra of PHO and PHO-Cl Samples
PHO sample 221 sample 191
carbon 1H 13C 1H 13C 1H 13C 0.87-0.92 0.96-1.15 11.00 20.85 -CH3 0.87-0.92 13.98 20.87 1.24 23.14 25.47 25.48 22.51 1.18-1.68 1.70-1.78 2.07-2.25 30.87 -CH2-(side 1.15-1.35 24.73 31.45 1.63-1.83 36.27 chains) 1.47-1.64 31.52 33.67 2.19-2.47 33.74 35.89 -CH2-CO2- 2.58 39.10 2.66 39.01 2.77 38.99 40.81 41.73 41.40 -CH2Cl 3.49-3.75 44.44 3.30-3.84 44.70 44.92 48.00 48.14 54.39 58.04 60.83 61.43 63.32 64.29 68.44 55.60 58.51 -CHCl- 4.06-4.55 59.80 4.06-4.74 65.15 66.75 68.34 -OCH- 5.20 70.85 5.22, 5.41 70.47 5.45 70.20 -CHCl2 5.96 72.29 -CCl2- 90.10 -CdO 169.43 169.07 168.66
Figure 5. DSC thermograms of the chlorinated polyesters (PHB-Cl: samples 211, 25, and 24; PHO-Cl: samples 191 and 221).
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 cross-linked polymer and dried under vacuum.
The1H 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
corre-sponding 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 monosub-stituted 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 the13C 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-1belong to water bands36due
to the hydroscopic natures of S-sulfate salts. FTIR absor-bances of S-sulfate salt are given as 1175, 1205, 1640, and 3450 cm-1in the literature.34The 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 Figure 6. 200 MHz1H NMR spectrum of PHAN+R
3recorded in CDCl3.
Table 4. Results, Conditions, and Solubility Properties of Quaternization, Friedel-Crafts, and Thiosulfate Reactions of PHA-Cl Samples
PHA reagents solvent solubilityb
run no. sample amount (g) type amount type volume, mL temp,°C yield (g) MeOH CHCl3 ether THF
N1 191 0.50 N(Et)3 2 mL DMF 10 22 0.63 s s s s N2 191 0.15 N(Et)3 0.5 mL CH2Cl2 15 3 0.16 s s s s N3 191 0.15 N(EtOH)3 0.5 mL DMF 5 22 0.18 s s s s N4 221 0.50 N(Et)3 2 mL DMF 10 22 0.60 s s s s N5 232 0.20 N(Et)3 3 mL DMF 3 60 0.22 s s s s Ph1a 221 0.15 AlCl 3 0.1 g benzene 25 reflux 0.23 ns s s s
Ph2 232 0.30 AlCl3 0.1 g benzene 40 reflux 0.40 ns s s s
S1 191 0.45 Na2S2O3‚5H2O 0.5 g acetone 15 reflux 0.71 ns s s s
S2 232 0.20 Na2S2O3‚5H2O 0.5 g acetone 20 reflux 0.27 ns s s s
solubilityb
starting polyesters MeOH CHCl3 ether THF
PHB ns s ns ns
PHO ns s ns s
PHB-Cl ns s ns ss
PHO-Cl ns s s ss
4) were Mn) 16100 and Mw/Mn) 1.6, respectively, while Mnof precursor 191 was 25600 Da.
FTIR Spectra. Characteristic FTIR spectra of the chlo-rinated 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:
There was a good agreement between the results of the Figure 8. FTIR spectra of (a) PHO-Cl (sample 221), (b) PHAN+R3,
and (c) PHA S.
Figure 7. 200 MHz1H NMR spectra of (a) PHB-Ph and (b) PHO-Ph recorded in CDCl 3.
wt. % of Cl in PHB-Cl )
(mol % of HCB)× 35.5
(mol % of HB)× 86 + (mol % of HCB) × 120.5
chlorine content obtained from the C, H analysis and 1H
NMR spectra. As typical examples, compare the Cl contents of the samples 232 and 211 in Table 5.
Acknowledgment. This work was financially supported by Zonguldak Karaelmas University Research Fund and TU¨ BI˙TAK/National Science Foundation-INT 997 4598 TBAG-U/1.
References and Notes
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Table 5. Elemental Analysis (C, H) Results of the Modified PHAs (wt %) calculated found entrya C H C H Cl Clb PHB 55.80 7.03 PHB-Cl 232 50.88 6.50 5.45 5.47 211 48.15 6.14 8.54 8.34 25 41.00 4.62 17.21 24 34.81 4.21 23.81 PHO 67.57 9.92 221 43.61 5.80 28.09 191 34.84 3.57 39.09 PHA-S S1 26.78 3.50 S2 43.14 5.71 PHA-N+R3 N1 43.41 5.47 N2 39.18 4.90 N5 43.01 5.60
