Characterization of Microbial Polythioesters: Physical Properties of

Biomacromolecules 2002, 3, 159-166

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Characterization of Microbial Polythioesters: Physical Properties of Novel Copolymers Synthesized by Ralstonia eutropha Tina Lu¨tke-Eversloh,† Jumpei Kawada,‡ Robert H. Marchessault,*,‡ and Alexander Steinbu¨chel*,† Institut fu¨r Mikrobiologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstrasse 3, D-48149 Mu¨nster, Germany, and Department of Chemistry, Mc Gill University, 3420 University Street, Montreal, Quebec H3A 2A7, Canada Received August 27, 2001; Revised Manuscript Received October 18, 2001

Various samples of polythioesters with different contents of 3-mercaptopropionic acid (3MP) or 3-mercaptobutyric acid (3MB) as one comonomer and with 3-hydroxybutyric acid (3HB) as the second constituent were produced by cultivating cells of Ralstonia eutropha strain H16 in mineral salts medium containing 3MP or 3MB plus gluconate as carbon sources. Fermentations were done also at the 30-L scale. The various samples were cast as films from chloroform and the following were recorded: melting point, solid-state NMR, X-ray diffraction. The copolyester poly(3HB-co-3MP) displayed mutiple melting peaks corresponding to separate phases rich in 3MP and 3HB. The copolyester poly(3HB-co-3MB) displayed very low crystallinity and melting points higher than that of poly(3HB) when the 3HB content was 40% or less. Introduction Polyhydroxyalkanoic acids (PHA) are a diverse class of bacterial polyesters which consist of hydroxyalkanoic acids linked by oxoester bonds. Poly(3-hydroxybutyric acid), poly(3HB), was the first example of PHAs detected almost 80 years ago.1 This and other polyesters occur in many bacteria as carbon reserve and energy storage compounds and are deposited as insoluble cytoplasmic inclusions in the cells.2,3 Polymerization is catalyzed by PHA synthases which use the coenzyme A thioesters of the hydroxyalkanoic acids as substrates.4,5 A large variety of different PHAs has been obtained;6 some of them might be used in the future for many applications in various areas due to their biodegradability7 and due to the perspective for biotechnological production. Some of them can now be produced from renewable resources by fermentation8 or in the future from carbon dioxide by agriculture using transgenic crops.9 This will depend on the success of metabolic engineering and the design of suitable pathways in the respective production organisms.10,11 The properties of various PHAs, in particular those of the homopolyester poly(3HB), the copolyester of 3HB and 3-hydroxyvaleric acid, poly(3HB-co-3HV), and of PHAs consisting of medium-chain-length 3-hydroxyalkanoic acids, poly(3HAMCL), have been studied in detail in the past.12 Recently, we detected a new class of biopolymer that was also synthesized by the PHA synthase. When the Gram* To whom correspondence should be addressed. R. H. Marchessault: tel, (514) 398-6276; fax, (514) 398-7249; e-mail, [email protected] (regarding physical properties). A. Steinbu¨chel: tel, + 49 (251) 8339821; fax, + 49 (251) 8338388; e-mail, [email protected] (regarding biological aspects). † Westfa ¨ lische Wilhelms-Universita¨t Mu¨nster. ‡ Mc Gill University.

Figure 1. Structural formula of poly(3HB-co-3MP) (a) and poly(3HBco-3MB) (b).

negative bacterium Ralstonia eutropha was cultivated in the presence of 3-mercaptoalkanoic acids or 3,3’-thiodipropionic acid (TDP), copolymers consisting of 3-mercaptoalkanoic acids and 3HB were synthesized. The constituents were linked by thioester as well as by oxoester bonds. So far, 3-mercaptopropionic acid (3MP)13 and 3-mercaptobutyric acid (3MB)14 have been described as constituents of these novel polythioesters (Figure 1). There were also efforts in the past to synthesize related polymers chemically.15 Shortly after the first report13 on these novel biopolymers was published, it was suggested by others that polythioesters may have some useful applications in medicine.16,17 The presence of thioester linkages in these mixed poly(thiooxo)esters will certainly effect the physical and material properties as well as the biodegradability of these polymers. Moreover, the release of inorganic sulfur compounds during biodegradation might have an effect on the biosphere in which these polymers are present. The aim of this study was to produce sufficient amounts of representatives of these novel polythioesters in order to reveal some physical properties of these polymers. In particular: X-ray diffraction, thermal analysis, and 13C solid-

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Biomacromolecules, Vol. 3, No. 1, 2002

Table 1. Chemical Formula, Crystal Information, Melting Temperature (Tm) and Reference of Polymers18,19

state NMR have provided the benchmark characteristics which distinguish solid PHAs from each other. When chemical analysis indicates that copolyesters are present, the microstructure of the chain as derived from high-resolution NMR is an important characteristic. These biopolyesters are of interest because sulfur-containing polymers examined over the past decades provide improved properties compared to oxygen equivalent analogues. For example, when sulfur atoms are present in the backbone, the melting temperature is higher than that of the polymer with equivalent oxygen atoms. Two well-known polyether examples are shown in Table 1. Materials and Methods Bacterial Strain and Culture Conditions. R. eutropha H16 (DSM 428) was used for this study. This strain was cultivated in nutrient broth (NB) growth medium or in mineral salts medium (MSM)25 at 30 °C in Erlenmeyer flasks under aerobic conditions on a rotary shaker with 130 rpm. To promote PHA accumulation, the ammonia concentration was reduced to 0.05% (w/v). Carbon sources were added from filter-sterilized stock solutions at the concentrations indicated in the text. Sodium gluconate or fructose was used as carbon source for growth. 3-Mercaptopropionic acid (3MP) or 3,3′-thiodipropionic acid (TDP) was used as a second, sulfur-containing carbon source to produce poly(3HB-co-3MP).13 3-Mercaptobutyric acid (3MB) was used as substrate for the biosynthesis of poly(3HB-co-3MB).14 Fed-batch cultivation of R. eutropha H16 at a 28-L scale was performed in a stirred (at 200-400 rpm) and aerated (15-20 L min-1) 30-L stainless-steel fermenter (Biostat UD30, B. Braun, Biotech International, Melsungen, Germany). Fermentations were carried out in MSM, and the pH was adjusted to 7.0. Analysis of Ammonium and Carbon Sources. The ammonia content in the culture broth was determined with a type 15 230 3000 gas-sensitive electrode (Mettler Toledo GmbH, Steinbach/Ts, Germany) according to the instructions provided by the manufacturer. Concentrations of gluconate and TDP in the culture supernatant were determined simultaneously by ion exchange

Lu¨tke-Eversloh et al.

chromatography using an HPLC apparatus (Knauer GmbH, Berlin, Germany). Supernatants were mixed (1:1, v/v) with sulfuric acid (5%, v/v), incubated at 70 °C for 3 h, and centrifuged at 13 000 rpm (Biofuge pico, Heraeus, Osterode, Germany) for 20 min before subjecting 10 µL to HPLC analysis. Degassed 6.5 mM sulfuric acid at a flow rate of 0.5 mL/min (L-6200 Gradienten Pumpe Lichrograph, Merck, Darmstadt, Germany) was used as the mobile phase. Separation was achieved in an Aminex ion-exclusion column (HPX87H, 300 × 7.8 mm, Biorad, Richmond, U.S.A.) at 65 °C (T-6300 column oven, Merck, Darmstadt, Germany). Detection was done using an RI detector (RI-71, Merck, Darmstadt, Germany), and data were evaluated by an integrator (Chromatopac C-R3A, Shimadzu, Duisburg, Germany). Polymer Isolation from Lyophilized Cells. Poly(3HBco-3MP) and poly(3HB-co-3MB) were extracted from lyophilized cells with chloroform, filtered, precipitated in 10 volumes of ethanol, and dried under a constant air stream. To obtain highly purified polymer, the precipitation procedure was repeated at least three times. GC/MS Analysis. The polyester content was determined by methanolysis of 5-7 mg of lyophilized cells in the presence of sulfuric acid, and the resulting methyl esters were characterized by gas chromatography.26,27 For molecular analysis of the methyl esters, a coupled gas chromatography/ mass spectrometry (GC/MS) was performed using a HP 6890 gas chromatograph equipped with a model 5973 mass selective detector (Hewlett-Packard, Waldbronn, Germany). The obtained mass spectra were compared with the NIST ′98 Mass Spectral Library with Windows Search Program Version 1.6, National Institute of Standards and Technology (U.S. Department of Commerce). Elemental Sulfur Analysis. Sulfur analysis was performed by the Mikroanalytisches Labor Beller (Go¨ttingen, Germany) according to the method of Grote and Krekeler (DIN 51768). Molecular Mass Analysis. The molecular masses of purified polyesters were estimated by gel-permeation chromatography (GPC) relative to polystyrene standards. Analysis was performed on four Styragel columns (HR 3, HR 4, HR 5, HR 6) connected in line in a Waters GPC apparatus (Waters, Milford, MA). Samples were eluted with chloroform at a flow rate of 1.0 mL/min and at 35 °C, and the eluted compounds were monitored by a Waters 410 differential refractometer. Polydispersity and the number average (Mn) and weight average (Mw) molar masses were calculated by using the Millenium Chromatography Manager GPC software (Waters, Milford, MA). X-ray Diffraction Analysis. X-ray diffraction data were collected by using a Rigaku D/Max 2400 diffractometer operated at 40 kV and 160 mA at room atmosphere. A copper X-ray anode was used with graphite monochromator, which provided Cu KR radiation. Thermal Analysis. All samples were pretreated as follows: first, the samples were heated from room temperature to 185 °C by 10 °C/min ramping; second, they were rapidly quenched from 185 °C to room temperature by 80 °C/min ramping and they were kept at room temperature for 10 min. After that, the samples were analyzed by a differential

Physical Characterization of Biopolythioesters

Biomacromolecules, Vol. 3, No. 1, 2002 161

Figure 2. Time course of a fed-batch fermentation of R. eutropha in a 30-L scale. The cells were cultivated for 48 h in mineral salts medium containing 1.5% (w/v) gluconate and 0.35% (w/v) TDP. The growth curve (OD600), concentrations of ammonium, gluconate, and TDP in the culture medium, the polymer content of the cells, and the 3MP content of the polymer are shown. During cultivation, NH4Cl and sodium gluconate were fed as indicated by the arrows. During the second part of the exponential growth phase, TDP was added continuously to a concentration up to 1.2% (w/v).

scanning calorimetry (DSC) instrument (Perkin-Elmer Pyris 1) with ramping at 10 °C/min. Polarized Microscopy. The sample was heated at 190 °C for 5 min. The melted sample was then cooled for 80 min to room temperature, and micrographs were recorded using a polarization microscope, Nikon MICROPHOT-FXA. 13C Cross-Polarization/Magic Angle Spinning NMR Spectroscopic Analysis. Cross-polarization/magic angle spinning (CP/MAS) NMR spectra were recorded by a Chemagnetics CMX-300 instrument operating at 75.4 MHz for 13C. Samples were packed in 7.5 mm PENCIL rotors and spun at 4000 Hz. Transmission Electron Microscopy. Single crystals of sample 2 were grown by a method modified from that of Nobes et al.28 The sample (9.5 mg) was dissolved in chloroform solution (25 mL), heated to 60 °C, and kept for 20 min. Ethanol (75 mL) heated at 60 °C was added to the solution, and the mixture was kept in a Dewar flask for 1 week until single crystals formed. Drops of poly(3HB-co3MP) single crystals suspended in excess methanol or water were deposited on carbon coated grids and allowed to dry. The grids of single crystals suspended in methanol were then

shadowed with Pt at an angle of 25° using a High Vacuum Coating Unit BAE 301 (Balzers AG, Liechtenstein). The grids were examined with a JEOL 2000FX transmission electron microscope operating at 80 kV for imaging. Photomicrographs were recorded electronically. Results Biosynthesis of Polythioesters. For synthesis of poly(3HB-co-3MP) (Figure 1a), R. eutropha was cultivated in a 30-L fermenter in order to produce suitable amounts of this polythioester biotechnologically. Fed-batch fermentations were performed in two stages: in the first stage sodium gluconate and ammonium chloride were provided to allow the cells to grow to high densities, whereas in the second stage TDP or 3MP was added for poly(3HB-co-3MP) accumulation. The time course of a typical fed-batch fermentation, from which cells polymer sample 10 was isolated, is shown in Figure 2, indicating a continuous increase of the polymer content of the cells and of the molar fraction of 3MP of the polymer accumulated in the cells, in the second stage. Approximately 330 g of dry cell matter

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Table 2. Polymer Samples Used for This Studya polymer composition (mol %)

sample no.

type of polymer

sulfur content of polymer (wt %)

3HB

3MP

1 2 3 4 5 6 7 8 9 10 11 12 13

poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MP) poly(3HB-co-3MB) poly(3HB-co-3MB) poly(3HB-co-3MB)

9.96 12.88 15.67 3.81 9.28 14.89 5.84 3.43 2.20 4.17 11.65 20.52 23.69

73.1 65.1 57.5 89.7 74.9 59.6 84.2 92.8 95.4 91.3 66.8 38.5 27.8

26.9 34.9 42.5 10.3 25.1 40.4 15.8 7.2 4.6 8.7

3MB

Mw of polymer

Mw/Mn of polymer

33.2 61.5 72.2

490 000 1 120 000 790 000 886 000 132 000 190 000 470 000 715 000 633 000 749 000 790 000 563 000 898 000

4.2 1.1 7.0 1.2 2.1 2.9 3.5 1.8 2.7 3.6 2.1 2.0 1.9

a Poly(3HB-co-3MP) and poly(3HB-co-3MB) were synthesized by R. eutropha.13,14 The elemental sulfur content was analyzed by the method of Grote and Kerkeler (DIN 51768) and the derived molar fraction of 3MP and 3MB, respectively, were calculated. The weight average molecular mass (Mw) and polydispersity (Mw/Mn) of the polymers were estimated by GPC relative to polystyrene standards

Figure 3. X-ray powder traces of polymer samples. The traces are numbered according to Table 2.

and up to 70 g of highly purified polymer could be obtained from an experiment done at the 30-L scale. Due to the poor availability of 3MB, poly(3HB-co-3MB) (Figure 1b) was only synthesized by R. eutropha at a 500 mL scale in 2-L Erlenmeyer flasks, and polymer samples were obtained in lower yields.14 In this study, 13 samples of 3MP and 3MB containing polymers were obtained from different cultivations of R. eutropha. Whereas in poly(3HB-co-3MP) the molar fraction of 3MP varried between 4.6 and 42.5%, the molar fraction of 3MB in the poly(3HB-co-3MB) samples varried between 33.2 and 72.2 mol %. The molecular weights were usually high, exceeding 500 kDa; only two samples of poly(3HBco-3MP) (samples 5 and 6) exhibited much lower molecular weights. Table 2 summarizes the polymer compositions, molecular weights, and polydispersity indices of the copolymers, which were further analyzed as described below. According to a recent study,29 the relative molecular weights revealed in this study by GPC must be corrected by a factor of 0.7 to correspond to the absolute molecular weights. X-ray Diffraction Analysis. X-ray diffraction powder patterns of samples 2, 3, 6, 9, 11, and 12 are shown in Figure 3. The X-ray pattern of sample 9 is in good agreement with that of pure poly(3HB). However, high molar 3MP samples 2, 3, and 6 showed a sharp peak at 21.3°, while poly(3hydroxypropionate), poly(3HP), does not show the diffraction

peak at 21.3°.30 Therefore, a significant difference is detectable in the diffraction patterns of the copolymer poly(3HBco-3MP) containing thioester linkages as compared to poly(3HB) but not due to a separate poly(3HP) phase. The 3MP units are probably randomly distributed in the polymer chain and hinder crystallite size development (cf. NMR data below). Samples 11 and 12 also showed X-ray patterns which are similar to that of poly(3HB). As for high molar 3MB the samples were elastic, but the crystallinity was poor and sample 13 is almost completely noncrystalline. Also sample 13 could be cold-stretched (200%) but did not yield an oriented fiber diagram. The lack of crystallinity in sample 13 is truly surprising since the content of 3MB was enough to produce a separate crystalline phase of poly(3HB) as in samples 11 and 12, which had a poly(3HB) phase. According to the research on poly(ethylene oxide), PEO, and poly(ethylene sulfide), PES,22 there is a significant difference in the molecular conformations of PEO and PES, although both polymers have similar chemical structures except for the oxygen and sulfur atoms. The reason for the different conformations may be the different bond length of C-O (1.43 Å) vs C-S (1.815 Å) and the van der Waals radii of the O (1.52 Å) vs S (1.85 Å) atoms. Thus, the molecular conformations of poly(3MP), poly(3HP), and poly(3MB) compared to poly(3HB) are probably different. Sample 9 with its low content of 3MP crystallizes predominantly as poly(3HB). Thermal Analysis. Examples of DSC results are shown in Figure 4, and Table 3 summarizes crystalization temperatures (Tc) and melting points (Tm). Some samples having high sulfur content showed two or three melting endotherms as shown in Figure 4a. Other samples, including low sulfur content, had only one Tm, as in Figure 4b, as usually found for poly(3HB). Sample 8 was the only exception. Tm1 is associated with 3MP-rich phases (Tm is 121 °C for poly(3HP))31 and Tm2 derives from poorly organized domains of 3HB and 3MP copolymer. Tm3 is assigned to the Tm of poly(3HB) (usually about 175 °C). All samples showed the Tm3 peak at around 165 °C depressed by the presence of



Table 3. Thermal Analysis of Poly(3HB-co-3MP), Poly(3HB-co-3MB), and Reference Poly(3HB) sample no. 1 2 3 4 5c 6 7 8 9 10 11 12 13 poly(3HB) d

polymer composition (mol %) 3HB 73.1 65.1 57.5 89.7 74.9 59.6 84.2 92.8 95.4 91.3 66.8 38.5 27.8 100

3MP

3MB

Tg (°C) -3.3

26.9 34.9 42.5 10.3 25.1 40.4 15.8 7.2 4.6 8.7

-1.7

33.2 61.5 72.2 4.0

Tc (°C)

Tm1 (°C)

Tm2 (°C)

Tm3 (°C)

62.6 50.3 64.4 b 71.0 56.9 59.1 52.2 52.3 53.3

(102.2)a

137.5 132.6

(153.9)a 168.6 159.2 173.2 163.9 164.9 163.2 163.2 164.2 162.9 168.6 225d

45.3

111.6 111.1

150.2 123.4 (155.0)a 151.3

112.2

171.8

a Not well-defined endotherm. b Sample 4 crystallized during quenching, so that it did not show the T peak. c Data were recorded before quenching. c Data were recorded by DTA.

Figure 4. DSC thermograms of sample 2 (a) and sample 10 (b).

comonomer. The crystallization exotherm (Tc) was from 50 to 71 °C with the higher values usually associated with high sulfur content. In addition, all Tc values of the samples are higher than that for the poly(3HB) reference sample in Table 3 because their copolymer composition hinders crystallization. As shown in Figure 4, sample 10 showed a narrow Tm melting range, like pure poly(3HB), while sample 2 had a wide melting point range, since the 3MP content is quite different between samples2 and 10. Sample 2 had a high 3MP content and it showed three Tm values, suggesting that cocrystallization may happen. By contrast, sample 10 with a lower 3MP content showed only one Tm, like pure poly(3HB). The correlation of the 3MP content of poly(3HB-co-3MP) samples to Tc and Tm3 values in Figure 5 shows large fluctuations which suggests that microstructure varies widely as a function of 3MP content in the product. Another explanation is the sensitivity of crystallization to aging phenomena in these samples. Because of instrument limitations, only two Tg values were recorded, both of which are 6-8 °C below the reported value for pure poly(3HB).12

Figure 5. Relation of the 3MP content of poly(3HB-co-3MP) to the crystallization temperature (Tc) (a) and to the melting temperature (Tm3) (b).

Polarized Microscopy. The “as received” solvent cast films did not show birefringence in the polarization microscope. When sample 1 was melted at 190 °C for 5 min and cooled to room temperature, spherulites were detected. Two kinds of spherulites were observed as shown in Figure 6: One is the usual spherulite from homopolymers (concentric rings) and the other is dendritic. From the results of thermal analysis, it seems that these samples do not decompose under 200 °C. However, they may depolymerize sufficiently to form a low molecular weight fraction giving rise to the needlelike clusters. 13C-CP/MAS NMR Spectroscopic Analysis. The crosspolarization/magic angle spinning, CP/MAS NMR, spectra of samples 2 and 8 are shown in Figure 7. The spectrum (Figure 7a) of sample 2 shows narrow resonances: 21.5, 43.0, 68.7, and 170.0 ppm due to the 3HB units and three of lesser amplitude at 23.9, 46.2, and 198.9 ppm due to the 3MP units. All peaks are consistent with both the 3HB and

164



Figure 6. Spherulitic morphology of sample 1.

Figure 8. 13 (b).

Figure 7. 8 (b).

13C

CP/MAS NMR spectrum of sample 2 (a) and sample

3MP repeat units being in well-ordered domains. However, a one-pulse Bloch decay spectrum (not shown) suggested that noncrystalline domains are also present and 3MP repeat units are probably in these domains. The CP/MAS spectrum (Figure 7b) of sample 8 is basically the same as that of crystalline poly(3HB). However, the CdO resonance (about 200 ppm) of 3MP is barely detectable. This implies that the 3MP in this low 3MP content sample is in a disordered mobile state; hence, it does not register in the CP/MAS spectrum. In Figure 8a for sample 11, the larger amplitude peaks are attributed mainly to a 3HB crystalline phase while the smaller amplitude peaks are attributed to 3MB repeating units in a copolymeric phase. Sample 13, Figure 8b, has the 3HB and 3MB peaks inverted in amplitude in view of the different composition. However, the breadths of the peaks in Figure 8b are all greater than those seen in the spectrum of sample 11 (Figure 8a). This is a sign of disorder in keeping with our X-ray diffraction observations on the same samples. This result suggests that the poly(3HB) phase in sample 13 is cocrystallizing with poly(3MB) in the solid sate. To examine the textural homogeneity of the CHCl3 cast film used in this CP/MAS analysis, the spectrum for sample 11 in Figure 8a was deconvoluted and the 3MB content was derived. The value obtained was 36%, which is in reasonable agreement with the data in Table 2. A subsequent measuring of 1H T1F values to test if the crystalline and noncrstalline

13C

CP/MAS NMR spectrum of sample 11 (a) and sample

phases were completely miscible indicated that they were not. The two components reside in separate domains of diameter greater than ∼3-6 nm. Transmission Electron Microscopy. Poorly defined lamellar single crystals of sample 2 showed two types of morphology as in Figure 9: one is a typical lamellar structure, the other is rod type. The typical lamellar structure is similar to poly(3HB). However, the rod type was also detected and as of now we cannot associate it with a given composition or polymorph. Discussion Sulfur is an abundant element on earth and an essential element for all organisms, which is used in various oxidized and reduced forms by the living matter. In particular, microorganisms have developed various strategies of using inorganic sulfur compounds in energy metabolism. Moreover, it is often found in certain bacteria in the form of liquid sulfur globules. A notable discovery concerning sulfur in bacteria was the report on sulfur-containing polymers from bacteria which contain thioester linkages.13 These polymers were in the family of PHA, which had never been reported to contain sulfur in the backbone chain. The sulfur is in the polymer backbone replacing the ester oxygen. The reported polymers were copolymers of 3-hydroxybutyrate and 3-mercaptopropionate which, however, were not random copolymers but were described as slightly “blocky” on the basis of NMR evidence.13 Exploring the field of polymers in general, one can compare polyethylene oxide and polyethylene sulfide, which are identical macromolecules except for the replacement of sulfur by oxygen or vice versa. Of note is the fact that the



account for the multiple melting endotherms. It is easy to predict that major differences in mechanical properties between polythioesters and PHA will be found based on our preliminary observations of relaxation properties. Biotechnologically produced sulfur-containing polymers might therefore provide novel materials, e.g., for medical applications.16,17 Nomenclature PHA: polyhydroxyalkanoate PHB: poly(3-hydroxybutyrate) TDP: 3,3′-thiodipropionic acid 3MP: 3-mercaptopropionic acid 3MB: 3-mercaptobutyric acid poly(3HB-co-3MP): poly(3-hydroxybutyrate-co-3-mercaptopropionate) poly(3HB-co-3MB): poly(3-hydroxybutyrate-co-3-mercaptobutyrate) Tg: glass transition temperature Tc: crystallization temperature Tm: melting temperature DSC: differential scanning calorimetry CP/MAS NMR: cross-polarization/magic angle spinning nuclear magnetic resonance

Figure 9. Single crystals of sample 2, which were suspended in water and observed without shadowing (a) and suspended in methanol and observed after shadowing with platinum (b).

melting point for polyethylene sulfide is nearly 100 °C higher than the oxygen-bearing macromolecule (cf. Table 1). While it may be true that some sulfur-containing polymers have a higher melting point than the equivalent oxygen-containing polymer, there is no evidence that the presence of a large amount of sulfur in these polythioesters has any particular effect on the melting point which cannot be explained in terms of copolymer depression of the melting point and crystalline disorder. One need homopolymers of these polythioesters to have benchmark references. Unfortunately, homopolythioesters of 3MP or 3MB cannot be produced biotechnologically at present. Samples 12 and 13 are intriguing because the former did show an unexpectedly high melting point (225 °C). Sample 13 which was almost completely amorphous is reminiscent of polysulfide rubber which undergoes sulfur bond interchange on heating, thus providing a stress relaxation mechanism which prevents crystallization on stretching.32 The latter observation was made on sample 13 in our experiments. In this study we have reported some physical properties of these linear thiopolyesters and compared them with those of conventional polyhydroxyalkanaotes. Specifically we have compared solid-state NMR, thermal properties, and X-ray diffraction as well as some crystalline morphology observations. The data from this study support the conclusion by Lu¨tke-Eversloh et al.13 that the polythioesters are true copolymers but with a nonrandom microstructure which may

Acknowledgment. We are grateful to Metabolix/Tepha Inc. for financial support, enabling the biotechnological production of polythioesters. We also thank Markus Po¨tter for performing GPC analysis of the polymer samples and Daniel Mergelkamp for performing some of the cultivations experiments. Solid-state NMR spectra were recorded and discussed with us by Dr. Fred Morin of the McGill Chemistry Department. Professor Musa Kamal of the McGill Chemistry Engineering Department provided DSC data. Financial support from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. References and Notes (1) Lemoigne, M. Bull. Soc. Chim. Biol. 1926, 8, 770-723. (2) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450-472. (3) Steinbu¨chel, A. Biomaterials; Byrom, D., Ed.; MacMillan Publishers: Basingstoke, U.K., 1991; pp 123-213. (4) Rehm, B. H. A.; Steinbu¨chel, A. Int. J. Biol. Macromol. 1999, 25, 3-19. (5) Steinbu¨chel, A.; Hein, S. AdV. Biochem. Eng. Biotechnol. 2001, 71, 81-123. (6) Steinbu¨chel, A.; Valentin, H. E. FEMS Microbiol. Lett. 1995, 128, 219-228. (7) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451-463. (8) Choi, J.; Lee, S. Y. Appl. Microbiol. Biotechnol. 1999, 51, 13-21. (9) Poirier, Y. AdV. Biochem. Eng. Biotechnol. 2001, 71, 209-240. (10) Madison, L. L.; Huisman, G. W. Microbiol. Mol. Biol. ReV. 1999, 63, 21-53. (11) Steinbu¨chel, A. Macromol. Biosci. 2001, 1, 1-24. (12) Hocking, P. J.; Marchessault, R. H. In Chemistry and technology of biodegradable polymers; Griffin, G. J. L., Ed.; Chapman and Hall: London, 1994; pp 48-96. (13) Lu¨tke-Eversloh, T.; Bergander, K.; Luftmann, H.; Steinbu¨chel, A. Microbiology 2001, 147, 11-19. (14) Lu¨tke-Eversloh, T.; Bergander, K.; Luftmann, H.; Steinbu¨chel, A. Biomacromolecules 2001, 2, 1061-1065. (15) Marvel, C. S.; Kotch, A. J. Am. Chem. Soc. 1951, 73, 1100-1102. (16) Anonymous, Microbiol. Today 2001, 28, 37. (17) Anonymous, Chem. Ind. 2001, issue No. 3, 64.

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Characterization of Microbial Polythioesters: Physical Properties of

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