Biomacromolecules 2003, 4, 1800-1804
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Synthesis of Poly(E-caprolactone)-b-Poly(γ-benzyl-L-glutamic acid) Block Copolymer Using Amino Organic Calcium Catalyst Guangzhuo Rong, Mingxiao Deng, Chao Deng, Zhaohui Tang, Longhai Piao, Xuesi Chen,* and Xiabin Jing State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China Received June 27, 2003; Revised Manuscript Received September 11, 2003
A biodegradable two block copolymer, poly(-caprolactone)-b- poly(γ-benzyl-L-glutamic acid) (PCL-PBLG) was synthesized successfully by ring-opening polymerization of N-carboxyanhydride of γ-benzyl-L-glutamate (BLG-NCA) with aminophenyl-terminated PCL as a macroinitiator. The aminophenethoxyl-terminated PCL was prepared via hydrogenation of a 4-nitrophenethoxyl-teminated PCL, which was novelly obtained from the polymerization of -caprolactone (CL) initiated by amino calcium 4-nitrobenzoxide. The structures of the block copolymer and its precursors from the initial step of PCL were confirmed and investigated by 1H NMR, FT-IR, GPC, and FT-ICRMS analyses and DSC measurements. Introduction During the last two or three decades, biodegradable polymers have found several important pharmaceutical and biomedical applications.1-2 Aliphatic polyesters based on hydroxyalkanoic acids, such as poly(lactic acid), poly(glycolic acid), poly(-caprolactone), and their copolymers, are well-known biodegradable materials. Because of their low immunogenicity and good biocompatibility, they are widely used and/or investigated in numerous medical and pharmaceutical applications.3-5 Inclusion of the functional side groups of -COOH in aspartic acid and glutamic acid and of -NH2 in lysine in polyesters can help to improve their affinity to proteins and cells or to covalently or ionically combine with drugs, antibodies, or DNA’s and thus may lead to breakthrough in the fields of targeting drug delivery and gene delivery. In addition, the presence of peptide bonds in the polymer backbone can modify the degradation pattern of the polymers, making them susceptible to peptidases. Synthetic poly(amino acids) can be rationally synthesized by means of ring-opening polymerization (ROP) of R-amino acid N-carboxyanhydrides (NCA) initiated by primary amine,6 and their biodegradation in mammalian tissues can be well controlled by their structure.7 The main potential medical applications for synthetic R-amino acid based polymers are bioresorbable sutures, screws, or plates and drug delivery systems (controlled release and targeting). There have been several papers on the studies of synthesis and characterization of polyester-co-poly(R-amino acid) block copolymer. Goshen had synthesized poly(L-lactide)b-poly(R-amino acid) by using endcapping method and also by using diethylzinc catalyst.8 Kricheldorf had synthesized various poly(R-amino acid)-b-poly(-caprolactone)-b-poly* To whom correspondence should be addressed. Phone: +86-4315262112. Fax: +86-431-5685653. E-mail:
[email protected].
(R-amino acid) A-B-A triblock copolymer by using 2, 2-dibutyl-2-stanna-1, 3-dioxepane as initiator.9 In this paper, we report a novel synthesis of poly(-caprolactone)-b-poly(γ-benzyl-L-glutamic acid) block copolymer using amino organic calcium initiator. The structures of the block copolymer and its precursors were confirmed by 1H NMR, IR, GPC, FT-ICRMS, and DSC. Experimental Section Materials. -Caprolactone (Aldrich) was dried over CaH2 for 1 week and distilled at reduced pressure prior to use. 4-Nitrophenethanol purchased from Aldrich was used without further treatment. Metal Ca was used without further treatment. N-Carboxyanhydride of γ-benzyl-L-glutamate (BLGNCA) was prepared according to the method of Daly.10 Pd/C(10%) was purchased from Chenzhou Xiangchen corporation in China and was used as received. Toluene, hexane, and tetrahydrofuran (THF) were dried by refluxing over Na metal under argon atmosphere and distilled immediately before use. Chloroform was refluxed over CaH2 and distilled under nitrogen. Measurements. FT-IR spectra were recorded on a BioRad Win-IR instrument. NMR spectra were measured by a Unity-300 NMR spectrometer at room temperature, with CDCl3 as solvent and TMS as internal reference. The GPC measurements were conducted at 35 °C with a Waters 410 GPC instrument with two Waters Styragel columns (HT6E, HT3) and a differential refractometer detector. The calibration of the columns was achieved with polystyrene standards. Tetrahydrofuran was used as eluent at a flow rate of 1.0 mL/min. DSC data were collected on a Perkin-Elmer apparatus (Pyris 1) at a rate of 10 °C/min. The FT-ICRMS experiment was carried out on a IonSpec 7.0T apparatus equipped with a YAG laser, and the sample was mixed with 2,5-dihydroxybenzoic acid (DHB, ppm concentration of sample in
10.1021/bm034208z CCC: $25.00 © 2003 American Chemical Society Published on Web 10/23/2003
Synthesis of PCL-PBLG
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Scheme 1. Synthesis of the PCL-PBLG Block Copolymer
acetonitrile/0.1%TFA 1:1 v/v) prior to loading on the sample stage for analysis, and the spectrum was recorded in the positive ion mode and one spectrum with one scan. Preparation of Calcium 4-Nitrophenethoxide Catalyst. Gaseous NH3 was purified over two columns filled with sodium sulfate and sodium hydroxide, respectively, to remove water and aldehyde impurities and was liquidized in a dry ice bath. 30 mL of liquidized NH3 was introduced into a dry flask containing 0.22 g of metal Ca, and the reaction was completed in 20 min. Then, 0.92 g of 4-nitrophenethanol was added into the reactor to continue the reaction in 30 min, and the excess NH3 was removed by raising the temperature from -40 °C to 100 °C. The obtained catalyst powder was preserved under nitrogen atmosphere. Preparation of Nitrophenyl-Terminated Poly(E-caprolactone), PCL-1. The polymerization was carried out in a nitrogen-filled and flame-dried glass reactor. 6.3 g of -caprolactone, 20 mL of toluene, and 1.22 g of calcium 4-nitrophenethoxide catalyst was introduced into the reactor in sequence, and the reactor was heated at 70 °C for 12 h. The product was cooled and then was precipitated into an excess of ethyl ether, washed several times with alcohol, and finally dried in a vacuum at room temperature for 24 h. Yield: 6.3 g (87.2%). Preparation of Aminophenyl-Terminated Poly(E-caprolactone), PCL-1A. A parr hydrogenation bottle was charged with 3.0 g of PCL-1, 40 mL of dry THF, and 0.3 g of palladium on charcoal (10% Pd). The bottle was pressurized with 1.0 MPa of hydrogen and allowed to be shaken at room temperature for 10 h. The flask was then carefully evacuated and filled with dry nitrogen. The solution was filtered and concentrated by rotary evaporation. The polymer was precipitated into diethyl ether and dried in a vacuum at room temperature for 24 h. Yield: 2.6 g (86.7%). Preparation of PCL-PBLG Diblock Copolymer. In a dry glass flask, 0.3 g of PCL-1A and 1.1 g of BLG-NCA
were dissolved in dried chloroform (20 mL). The ROP reaction mixture was stirred for 24 h at room temperature. Then, the mixture was precipitated with an excess of methanol under vigorous stirring and dried under vacuum at room temperature for 24 h. Yield: 0.9 g (74.0%). Results and Discussion It has been reported that the ROP of NCA can be initiated by primary amines.6 In this way, it is well-known that the initiator amine undergoes a nucleophilic addition to the C-5 carbonyl group of the NCA. Amino-terminated PCL is a desirable macromolecular initiator for the ROP of NCA. Because the weak aliphatic ester bonds in the PCL chain are easily broken down under strong reaction conditions such as strong acid or strong alkali, it is difficult to convert hydroxyl-terminated PCL into amino-terminated PCL by the usual methods. In our present study, we prepare the aminoterminated PCL by using an amino calcium catalyst with 4-nitrophenethanol as initiator. Furthermore, the amino organic calcium normally was a nontoxic catalyst, so that it has potential applications in medical and pharmaceutical fields. The PCL-PBLG diblock copolymer was successfully synthesized by ROP of BLG-NCA with the amino-terminated PCL, and its synthetic route is outlined in Scheme 1. The nitrophenyl-terminated PCL was synthesized with high conversion by amino organic calcium catalyst. The polymerization and possible mechanism of -caprolactone initiated by amino organic alkali metals has been reported in our previous work.11-13 The characteristics of the 4-nitrophenethoxy-initiated PCL are shown in Table 1. It was observed that molecular weights of the polymers are higher than the expected molecular weight. This is possibly attributed to the aggregation of the amino organic calcium initiator. The molecular weights and their polydispersity of nitrophenyl-terminated PCL were characterized by theoretical calculation, 1H NMR and GPC. Typical signals of both PCL
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Rong et al.
Table 1. Synthesis of Nitrophenyl-Terminated PCL Using Amino Calcium Catalyst polymer
M/I (mol)
Mna
Mnb
DPb
Mnc
polydispersity
PCL-1 PCL-2 PCL-3
10 19 24
1330 2400 2900
1660 3400 5260
13 29 45
2400 4030 5600
1.20 1.27 1.40
a
Calculated from M/I. b Calculated from 1H NMR. c Obtained from GPC.
Figure 3. FT-ICRMS spectrum of PCL-1A. Table 2. Effect of Hydrogenation polymer
Mna (before)
polydispersity (before)
Mna (after)
polydispersity (after)
PCL-1A PCL-2A PCL-3A
2400 4030 5600
1.20 1.27 1.40
2390 4460 6030
1.16 1.13 1.13
a
Figure 1. 1H NMR spectra of (A) nitrophenyl-terminated PCL-1, (B) aminophenyl-terminated PCL-1A.
Figure 2. IR spectra of (A) hydroxy-terminated PCL, (B) nitrophenylterminated PCL-1, (C) aminophenyl-terminated PCL-1A, and (D) PCLPBLG.
and nitrophenyl units were detected by 1H NMR as shown in Figure 1A. The FT-IR spectrum of nitrophenyl-terminated PCL was shown in Figure 2B. Compared to the FT-IR spectrum of the hydroxy-terminated PCL (Figure 2A), the absorptions at 1602 and 1522 cm-1 from the phenyl group and the peak at 1347 cm-1 belonging to the symmetric stretching modes νs of the nitro group were detected. The GPC traces of all polymers showed a unimodal polydistribution, which further indicated that the nitrophenylterminated PCL was successfully obtained. The nitro group of the polymer chains underwent facile reduction by Pd/C and H2 to yield the desired aminophenylterminated polymers.14 The significant upfield shift of the
Obtained from GPC.
“a protrons” from 8.18 to 7.01 ppm and “b protrons” from 7.38 to 6.64 ppm after hydrogenation was observed in the 1 H NMR spectrum (Figure 1B) of aminophenyl-terminated PCL-1A in comparison with that of nitrophenyl-terminated PCL-1 (Figure 1A). In the FT-IR spectrum (Figure 2C), two new absorptions appeared at 3456 and 3374 cm-1 (νas and νs of the amino group) and the peak at 1347 cm-1(νs of the nitro group) disappeared in comparison to Figure 2B, indicating that the nitro-group had been reduced to aminoPCL. The successful hydrogenation of the nitrophenyl end group was also confirmed by FT-ICRMS mass spectrometry (Figure 3). The masses of the polymer chains could be expressed as M ) 114.14n + 136.17 + 22.99, where 114.14 was the molecular weight of -caprolactone and 136.17 and 22.99 were the molecular weights of the 4-aminophenethanoxy group and the sodium cation, respectively. It means that the end group of the polymer chain was exactly the amino group. The data of Mn and polydispersity of aminoterminated PCL are shown in Table 2. Obviously, the molecular characteristic of the polymeric backbone was kept unchanged. In the present study, aminophenyl-terminated PCL was used as the macromolecular initiator for the ROP of NCA. The PCL-b-PBLG copolymer was purified by precipitating with an excess of methanol. The degree of polymerization (DP) and Mn (calculated from 1H NMR spectrum, Figure 2) of the block copolymers were shown in Table 3. The chain length of the PCL block was varied between 13 and 45, and the chain length of PBLG was varied between 31 and 60. The chain lengths of each block can be controlled by adjusting the amount of monomer CL and NCA and the ratio of monomer to initiator. The 1H NMR spectrum of the PCL-PBLG block copolymer is shown in Figure 4. Peaks at 7.26, 5.03, 3.93, 2.60, and 2.26 ppm are assigned to protons i, h, e, g, and f in the PBLG unit, respectively. Peaks at 4.06, 3.65, 2.30, 1.63, and 1.39 ppm corresponded to protons a, k, d, b, and c in the
Biomacromolecules, Vol. 4, No. 6, 2003 1803
Synthesis of PCL-PBLG Table 3. Molecular Weight Characteristics of the PCL-PBLG Block Copolymer PCL
PBLG
polymer
macroinitiator
Mna
DPb
DPa
Mna
PCL-PBLG1 PCL-PBLG2 PCL-PBLG3 PCL-PBLG4 PCL-PBLG5
PCL-1A PCL-1A PCL-2A PCL-2A PCL-3A
2390 2390 4460 4460 6030
22 43 21 41 52
31 55 31 61 60
6790 12040 6790 13360 13140
a
Calculated from 1H NMR. b Calculated from feed composition.
Figure 6. DSC curves of PCL and PCL-PBLG copolymers.
Figure 4.
1H
NMR spectrum of PCL-PBLG.
lymerization has been completed successfully and no homopolymerization occurred. However, this method could not be used to determine the molecular weights or the polydispersity indices of the block copolymer, because self-assembly of the diblock copolymer can exist.15 The DSC curves of PCL and PCL-PBLG were shown in Figure 6. An apparent melting temperature was 58-64 °C for a PCL homopolymer. The presence of PCL in copolymer PCL38-PBLG31 was reflected by the melting temperature at 59 °C. If the relative length of the PCL block became short, its Tm peak was weakly detectable or undetectable. The presence of the PBLG block was verified by a small peak with a typical low enthalpy change observed around 110 °C (TLC). This observed transition was irreversible and only occurred during the first heating run; it was attributed to an irreversible change from a 7/2 to an 18/5 R-helical conformation.16-17 At higher temperature, no other transition could be observed for PBLG until its degradation.18-20 Conclusion
Figure 5. GPC traces of (A) PCL-1, (B) PCL-1A, and (C) PCLPBLG1.
PCL unit, respectively. The existing of the peak at 3.65 ppm (characteristic of the end CH2OH of PCL) confirms that the amino group of macroinitiator initiates the polymerization of NCA, which is in accordance with the results of Goshen8 and Caillol.15 No additional peaks were detected in the spectrum, implying the high purity of the block copolymer prepared. The IR spectrum of PCL-PBLG is shown in Figure 2D. The absorption peak at 3292 cm-1 is assigned to the νNH stretch vibration of PBLG, and the peaks at 1653 cm-1 (νCO) and 1548 cm-1 (νCO-NH) are attributed to the amide group, indicating the formation of the polypeptide block. The GPC (Figure 5C) measurements show a sharp unimodal polydistribution. It further indicates that the copo-
The preliminary results show that the amino-terminated PCL synthesized by amino calcium 4-nitrophenethoxide is an effective macroinitiator for the ring opening polymerization of R-amino acid N-carbocyanhydrides. The controllable copolymerization of the amino-terminated PCL with NCA offered the possibility to produce a functional polymer with varying ratios of chain segments and functional groups. After catalytic hydrogenation of the PCL-PBLG diblock copolymer,21-22 the PCL-poly(L-glutamic acid) block copolymer can be prepared. This kind of block copolymer combines the characters of polyester and poly(amino acids). The copolymer will consist of hydrophilic/hydrophobic and functional groups, etc. The functional groups formed can be used to connect peptides, antibodies, and even DNA’s. Therefore, it is a promising biomedical material. Further investigations are undertaken. Acknowledgment. The authors are thankful to the National Natural Science Foundation of China and the “863” Project from the Ministry of Science and Technology of China for financial support to this work.
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References and Notes (1) Kricheldorf, H. R.; Kreiser-Saunders, I. Macromol. Symp. 1996, 103, 85. (2) Schroeter, J. Kunstoffe. 2000, 90, 1. (3) Holland, S. J.; Tighe, B. J.; Gould, P. L. J Controlled Release 1986, 4, 155. (4) Reed, A. M.; Gilding, D. K. Polymer 1981, 22, 494. (5) Gilding, D. K.; Reed, A. M. Polymer 1979, 20, 1459. (6) Blout, E. R.; Karison, R. H. J. Am. Chem. Soc. 1956, 78, 941. (7) Rypacek, F.; Pytela, J.; Kotva, R.; Skarda, V. Macromol. Symp. 1997, 123, 9. (8) Goshen, M.; Keel, H.; Ho¨cker, H. Macromol. Chem. Phys. 1995, 196, 3891. (9) Kricheldorf, H. R.; Hauser, K. Biomacromolecules 2001, 2, 1110. (10) Daly, W. H.; Poche´, D. Tetrahedron. Lett. 1988, 29, 5859. (11) Piao, L. H.; Deng, M. X.; Chen, X. S.; Jiang, L. S.; Jing, X. B. Polymer 2003, 44, 2331. (12) Piao, L. H.; Dai, Z. L.; Deng, M. X.; Chen, X. S.; Jing, X. B. Polymer 2003, 44, 2025.
Rong et al. (13) Tang, Z. H.; Chen, X. S.; Liang, Q. Z.; Bian, X. C.; Yang, L. X.; Piao, L. H.; Jing, X. B. J Polym. Sci. Part A: Polym. Chem. 2003, 41, 1934. (14) Carter, K. R.; Richter, R.; Kricheldorf, H. R.; Hedrick, J. L. Macromolecules 1997, 30, 6074. (15) Caillol, S.; Lecommandoux, S.; Mingotaud, A. F.; Schappacher, M.; Soum, A.; Bryson, N.; Meyrueix, R. Macromolecules 2003, 36, 1118. (16) Lecommandoux, S.; Achard, M. F.; Langenwalter, J. F.; Klok, H. A. Macromolecules 2001, 34, 9100. (17) Watanabe, J.; Uematsu, I. Polymer 1984, 25, 1711. (18) Gallot, B. Prog. Polym. Sci. 1996, 21, 1035. (19) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 1998, 280, 1427. (20) Klok, H. A.; Langenwalter, J. F.; Lecommandoux, S. Macromolecules 2000, 33, 7819. (21) Wang, D.; Feng, X. D. Macromolecules 1997, 30, 5688. (22) Deng, X. M.; Yao, J. R.; Yuan, M. L.; Li, X. H.; Xiong, C. D. Macromol. Chem. Phys. 2000, 201, 2371.
BM034208Z
