Biomacromolecules 2002, 3, 1057-1064
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One-Step Synthesis of Amphiphilic Diblock Copolymers from Bacterial Poly([R]-3-hydroxybutyric acid) Franc¸ ois Ravenelle and Robert H. Marchessault* Chemistry Department, McGill University, 3420 University Street, Montre´ al, Quebec, Canada H3A 2A7 Received April 17, 2002; Revised Manuscript Received May 31, 2002
Catalyzed transesterification in the melt is used to produce diblock copolymers of poly([R]-3-hydroxybutyric acid), PHB, and monomethoxy poly(ethylene glycol), mPEG, in a one-step process. Bacterial PHB of high molecular weight is depolymerized by consecutive and partly simultaneous reactions: pyrolysis and transesterification. The formation of diblocks is accomplished by the nucleophilic attack from the hydroxyl end-group of the mPEG catalyzed by bis(2-ethylhexanoate) tin. The resulting diblock copolymers are amphiphilic and self-assemble into sterically stabilized colloidal suspensions of PHB crystalline lamellae. Introduction Poly([R]-3-hydroxybutyric acid), PHB, is a biopolyester produced by many microorganisms as an osmotically neutral carbon reserve.1 This polymer is biocompatible and biodegradable, which makes it attractive for use in biomedical materials such as surgical pins and drug carriers. As produced in bacteria, PHB has a relatively high molecular weight (Mw ≈ 200 000 to 1 000 000 g/mol), which is unsuitable for molecular design of specialty polymers such as amphiphilic block copolymers, e.g., with poly(ethylene glycol), PEG. For such applications, reactivity requirements need more manageable molecular weights, around 1000 to 5000 g/mol. Methods such as acid2 and base hydrolysis3 as well as pyrolysis4 are frequently used to depolymerize the natural PHB. The oligomers thus obtained can be further modified using the carboxylic acid end group to create new functionality. Kumagai et al.6 and Shuai et al.7 synthesized PHB-PEGPHB triblock copolymers using ring-opening polymerization of [R,S]-β-butyrolactone using PEG-based macroinitiators. The resulting copolymers possessed atactic PHB segments with good solubility characteristics but lacking crystallinity as solids. In this study, bacterial PHB was used; hence, PHB segments are 100% isotactic and thus more crystalline and water insoluble. The implication of these properties for the diblock copolymers synthesized here will be discussed further. Synthesis of diblock copolymers from bacterial PHB and monomethoxy poly(ethylene glycol), mPEG, have also been reported using the dehydrating agent dicyclocarbodiimide (DCC) and catalyst (dimethylamino)pyridine (DMAP).8 However, this method results in low yields and long reaction times. PHB oligomers have also been grafted onto other natural polymers such as chitosan.9 Catalyzed transesterification is a green chemistry method, if used in the melt, not only to depolymerize the high molecular weight PHB but also to form a diblock copolymer * To whom correspondence should be addressed. E-mail: robert.
[email protected]. Tel: 1-514-398-6276. Fax: 1-514-398-7249.
Scheme 1. Catalyzed Transesterification Reaction Scheme between Bacterial PHB and mPEGa
a The pyrolysis reaction mechanism and products are shown elsewhere.4
if the nucleophile in the reaction is the hydroxyl and/or amine end group of another polymer (Scheme 1). Numerous transition metal catalysts are available for transesterification,10 and enzymatic catalysis11,12 has also been reported. Such a reaction has been performed using poly([D,L]lactide), PDLLA, and mPEG in toluene13 or in the melt, with14 or without catalyst,15 and the resulting diblock copolymers were used either to release medication such as Taxol or as substrate for tissue culture applications. Diblock copolymers of PHB and mPEG could also be used to encapsulate drugs and act as amphiphilic drug carriers in the form of micro- or nanoparticles or as frozen micelles. Our motivation for the process used in this study was for cleaner chemistry providing high reactivity and avoiding solvent use. The rapid one-step synthesis described in this paper combines chain-cleaving and chain-coupling reactions, i.e., pyrolysis and transesterification in the absence of solvent. The chronology of each reaction depends on their respective rates, controlled by the temperature of reaction chosen and time of catalyst addition. These parameters are also investigated. Experimental Section Materials. High molecular weight PHB (Mn ) 300 000; PD ) 2.0) was obtained from Imperial Chemicals Ltd., ICI, labeled as BIOPOL, reference number Bx-IRD. Monomethoxy poly(ethylene glycol), mPEG, 2000 and 5000, and bis(2ethylhexanoate) tin catalyst were purchased from SigmaAldrich Canada Ltd. and used as received.
10.1021/bm025553b CCC: $22.00 © 2002 American Chemical Society Published on Web 07/13/2002
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Biomacromolecules, Vol. 3, No. 5, 2002
Ravenelle and Marchessault
Figure 1. Full 1H NMR spectrum of PHB-block-mPEG.
Transesterification Reaction. In a typical experiment at 190 °C, a cylindrical glass reactor with magnetic stirrer is loaded with 500 mg of PHB and 500 mg of mPEG 2000. The reactor is then plunged into a preheated oil bath at 190 °C under vacuum until the melt viscosity is low enough to be stirred into a homogeneous mixture (a 15 min period). This step also ensures that all residual water present is driven out of the system. Under a flow of argon, 70 mg of liquid catalyst is added, and after 15 min of reaction the reactor is removed from the oil bath and cooled to room temperature yielding a waxy solid product corresponding to 95-98 wt % yield. This yield is calculated as the ratio of weight of product obtained (minus the amount of catalyst added) versus the weight of mPEG and PHB added at the start. Unreacted mPEG chains are removed from the product mixture by dialysis of a precipitated colloidal suspension. The method used is similar to that used by Zhang et al.:16 Typically, the product mixture is first dissolved in hot dimethylformamide (DMF), a good solvent for both blocks, and then the solution is cooled to room temperature and filtered if necessary. Under vigorous agitation, distilled water is slowly added until the final DMF concentration is approximately 10%. At this point, the product has formed a stable colloidal suspension. The suspension is then transferred to a Spectra/Por #4 regenerated cellulose membrane for dialysis (molecular weight cutoff of 12000-14000). After dialysis, the product is either a colloidal suspension or a precipitate or both, depending on the molecular weight distribution and size range of the PHB blocks. The suspension is then freeze-dried to a white or light yellow fluffy powder. Subsequent analyses involve X-ray powder diffraction, gel permeation chromatography (GPC), 1H and 13C CP/ MAS NMR, and differential scanning calorimetry (DSC). The total yield is now 55-77 wt % (weight of product/weight of PHB and mPEG at start). This method does not avoid the
presence of free PHB chains that will inevitably be cocrystallized in the self-assembled diblock copolymers. However, overall analysis of data shows that the amount is small. Gel Permeation Chromatography (GPC). GPC analysis was performed using a Waters pump and two Waters Styragel columns (HR3 and HR4) connected in series. An HP 1047 RI Hewlett-Packard refractive index detector was used at 35 °C and poly(ethylene glycol) standards were used for calibration. Chloroform was used as solvent, and the flow rate was set at 0.6 mL/min. Analyses lasted 40 min. These conditions permitted optimum chromatographic resolution in the molecular weight range of interest. Proton Nuclear Magnetic Resonance (1H NMR). 1H NMR spectra were recorded using a Varian Unity 300 or 400 MHz at room temperature. Unless otherwise mentioned in this paper, samples were dissolved in deuterated chloroform CDCl3 containing tetramethylsilane TMS. Pulses of 90° were used with a relaxation delay d1 of 1.5 s and 60-120 transients were recorded. 13C Cross-Polarization Magic Angle Spinning Nuclear Magnetic Resonance (13C CP/MAS NMR). 13C CP/MAS NMR spectra were recorded using a Chemagnetics CMX300 instrument operating at 75.34 MHz for the 13C nucleus. Spectra were accumulated at a spinning rate of 4 kHz. The cross-polarization contact time was varied from 1 to 6 ms, and a recycle delay of 2 s was used. X-ray Diffraction. X-ray diffraction powder patterns were recorded using a flat film camera with a Philips X-ray generator employing Ni-filtered Cu KR radiation generated at 40 kV and 20 mA. Measurements of d spacing were calibrated using NaF diffraction. Differential Scanning Calorimetry (DSC). DSC experiments were performed using a DSC Q1000 calorimeter from TA Instruments. Data analysis was done using the TA Universal Analysis 2000 Software, version 3.3B. Samples
Biomacromolecules, Vol. 3, No. 5, 2002 1059
Diblock Copolymers of Bacterial PHB and mPEG
Figure 2. 1H NMR record of pyrolysis and transesterification at 170 °C (SnO20). Table 1. Comparison of Transesterification and β-Elimination Rates at Different Temperatures sample
temp (°C)
rate of β-elimination (min-1)
rate of transesterification (min-1)
SnO14 SnO16 SnO20
190 180 170
40 × 10-5 8 × 10-5 (2-3) × 10-5
10 × 10-5 6 × 10-5 6 × 10-5
of mPEG were analyzed by the following process: ramp from 40 to 100 °C at 20 °C/min, ramp to -70 °C at 10 °C/ min, ramp to 100 °C at 20 °C/min. Diblock copolymer samples were analyzed using the following program: ramp from 40 to 170 °C at 20 °C/min, ramp to -50 °C at 10 °C/ min, ramp to 170 °C at 20 °C/min. Thus, all samples have the same thermal history, thermograms reporting melting points show only the last heating ramp of each analysis. Results and Discussion The rapid one-step synthesis of low molecular weight diblock copolymers of PHB and monomethoxy poly(ethylene glycol) 2000 and 5000 (mPEG) in the melt was undertaken using bis(2-ethylhexanoate) tin as transesterification catalyst. Transesterification allows for rapid depolymerization and functionalization yielding amphiphilic diblock copolymers. This process is faster than using condensation reactions between mPEG and PHB oligomers,8 and the yield is much higher and contamination of products by reagents and catalyst like DCC and DMAP is avoided. Ester-linked diblocks of PDLLA have been reported using transesterification, but characterization of the reaction products or their proof of structure was not detailed.13,14,15,18 To our knowledge, this reaction has never been reported using PHB. Like other poly-
3-hydroxyalkanoates (PHAs), PHB is thermally unstable at temperatures around its melting points where it undergoes a McLafferty rearrangement (β- or cis-elimination) via a sixmembered ring intermediate, which is accessible in β-hydroxypolyesters.4 Such thermal degradation results in the formation of crotonate end-groups,5 referring to crotonic acid’s structure (cf. Figure 1). Since transesterification in the melt generally necessitates temperatures at which PHB undergoes pyrolysis, there are two overlapping reactions depolymerizing PHB. To better understand the kinetics of the two overlapping reactions, test experiments were done at three different temperatures: 190, 180, and 170 °C. The rates of crotonate end-group and ester group production during the reaction were followed using integrations of 1H NMR resonances obtained from small aliquots removed from the reaction mixture at different times during the test experiments. The alkene protons from the crotonate end-group, produced by thermal degradation, have resonances at 5.8 and 6.9 ppm (Figure 1b,c) while the triplet at 4.2 ppm (Figure 1g) represents two protons from the newly formed ester bond between PHB and mPEG as a result of transesterification (Figure 1). Since the latter represents two protons, it has been normalized to one in order to make a clear comparison between the two reaction rates measured. Because the crotonate end-group’s methyl proton’s resonance is overlapping with another peak, most probably coming from the catalyst or residue, it was not possible to use that peak as reference before purification (ratio not 3:1:1 for a:b:c Figure 1, more like 3.8:1:1). Reported resonances are versus CDCl3. Ratios of the integrations of these different end groups versus the unchanging integration of the mPEG backbone region (3.4-3.8 ppm) allow for comparison of time progress (rate) of the two reactions as shown in Figure 2. The effect of temperature on the rate of the two different reactions is derived from plots as in Figure 2 and tabulated in Table 1. There were no significant rate changes for pyrolysis due to the addition of the catalyst for β-elimination, as can be seen in Figure 2, where two catalyst additions were performed at different times. Rates were calculated using a linear regression of plots built using different 1H NMR resonance integration ratios as discussed above (cf. Figure 2). From Table 1 we can conclude that the rate of β-elimination is more sensitive to temperature than transesterification. Only at 170 °C is the rate of transesterification higher than the rate of β-elimination. However, even at this temperature there is still depolymerization caused by thermal degradation. Accordingly, in the 170-190 °C temperature range and under our reaction conditions the making of diblock copolymers
Table 2. Molecular Weights of Diblocks Obtained with Different Reaction Times and Catalyst Amount at 180 °C
SnO5 SnO6 SnO7 SnO8 SnO9 a
PHB wt (mg)
mPEG 2000 wt (mg)
catalyst (mg)
T (°C)
time (min)
Mn of diblock (PDI) (GPC)
% yield
500 500 500 500 500
1750 1750 1750 1750 1750
50 50 50 10 30
180 180 180 180 180
60 30 15 60 60
2300 (1.1) 2400 (1.12) 2900 (1.13) 4700 (NA)a 2400 (NA)a
77 56 55
