A Convenient Method for the Synthesis of Amine-Terminated Poly

Methoxy PEO (MPEO, Mn = 2000) was obtained from Aldrich, and was treated by azeotropic distillation with toluene to get rid of the small amount of wat...
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Bioconjugate Chem. 2002, 13, 1159−1162

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A Convenient Method for the Synthesis of Amine-Terminated Poly(ethylene oxide) and Poly(E-caprolactone) Feng-Zhu Lu,† Xiang-Yuan Xiong,‡ Zi-Chen Li,*,‡ Fu-Sheng Du,‡ Bao-Yan Zhang,† and Fu-Mian Li‡ Department of Polymer Science & Engineering, College of Chemistry, Peking University, Beijing 100871, China, and Department of Chemistry, Northeastern University, Shenyang 110004, China. Received March 19, 2002; Revised Manuscript Received June 25, 2002

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of R-(N-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.

INTRODUCTION

Poly(ethylene oxide) (PEO) and poly(-caprolactone) (PCL) have found many applications as biomedical materials (1, 2). Chemical modification, especially endfunctionalization, is an important way to expand the applications of these polymers. Amine-terminated polymers are important intermediates for the synthesis of novel polymeric materials. They can react with molecules containing reactive groups such as acid chlorides, sulfonyl chlorides, acid anhydrides, and activated esters to conjugate other bioactive molecules at the end of the polymer chains (3). Furthermore, they can also be used as macroinitiators for the ring-opening polymerization of amino acid N-carboxy anhydrides (NCAs) to prepare block copolymers containing polypeptide segments (4). The first paper for the synthesis of amine-end capped polyester was reported by Teyssie et al. (5). In their report, diethylaluminum 12-bromododecyloxide was used as an initiator of CL, yielding a bromo-terminated PCL, which was then converted into an azide-terminated PCL by the treatment with an excess of sodium azide. An amine-terminated PCL was finally obtained after catalytic hydrogenation. Star-like PCLs with amino end groups have also been reported by this group by using the same approach (6). But the synthesis of amineterminated PLA by this method was demonstrated to be with side reactions (7). A similar approach has been reported by Kricheldorf et al. for the synthesis of amineterminated aliphatic polycarbonates (8). The difference is that a nitrophenyl group was introduced to the aluminum alkoxide as an active initiator of trimethylene carbonate (TMC), aminophenyl end-capped poly(TMC), was obtained after catalytic reduction. Other approaches to prepare amine-terminated polyesters are the chain end modification of the polymer precursor under mild conditions. This can be achieved by the in situ reaction of 4-nitrobenzoyl chloride with a macrocyclic PCL, resulting in the formation of a R,ω-diamine-terminated telechelic PCL after reduction (9). Recently, Yuan et al. (10) prepared an amine-terminated MPEO-PCL diblock poly* Corresponding author. Tel: +86-10-6275-7155. Fax: +8610-6275-1708. E-mail: [email protected]. † Northeastern University. ‡ Peking University.

mer from the hydroxyl-terminated polymer through a three-step reaction based on the chemistry as in the case of Teyssie’s work. There have been many reports on the preparation of amine-terminated PEO from hydroxyl-terminated PEO in the literature. The chemistry involved in this synthesis is to convert the hydroxyl groups into bromo, chloro, sulfonic, and aldehyde groups, followed by other chemical reactions leading to the formation of amino groups. For example, Buckmann and Johansson reduced the bromized PEO in alcohol using ammonia and hexadiamine, respectively (11). Sulfonic group end-capped PEO was transferred into amine-terminated PEO via a classical Gabriel process (12). The aldehyde group precursor of PEO was treated with NaCNBH3 and converted into an amino group (13). Besides the above strategies, an approach similar to that for the preparation of amineterminated PCL was carried out by treating the chloro precursor with NaN3- and Pd/C-catalyzed hydrogenation successively (14). Though amine-terminated PEO can now be commercially available, these methods are still being used for the preparation of amine-terminated PEO of different chain lengths. We tried another general procedure to obtain amineterminated PCL and PEO from the hydroxyl-terminated precursors. It involved the condensation of hydroxylterminated PEO and PCL with N-benzyloxycarbonyl amino acid and subsequent catalytic hydrogenation under mild conditions to liberate the amino groups. The purification procedure was easy. Yet, to the best of our knowledge, only one publication has appeared so far dealing with the synthesis of an amine-terminated PLA by a similar procedure (4b). Although the structure of the amino acid used is not the same as the polymer, the short chain length compared with PEO and PCL would not influence the properties of the main chain, but alternate their reactivities. This approach may be a universal synthetic route with a high potential utility for the preparation of amine-end capped polymers from hydroxyl-terminated polymer precursors. EXPERIMENTAL PROCEDURES

Materials. -Caprolactone (-CL) was purchased from Acros, dried over calcium hydride for 48 h at room

10.1021/bc025531m CCC: $22.00 © 2002 American Chemical Society Published on Web 08/14/2002

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temperature, and then distilled under reduced pressure. Al(OiPr)3 and 10% Pd/C were also from Acros; 10% Pd/C was used as received, and Al(OiPr)3 was distilled under reduced pressure prior to use. Methoxy PEO (MPEO, Mn ) 2000) was obtained from Aldrich, and was treated by azeotropic distillation with toluene to get rid of the small amount of water prior to use. N-Benzyloxycarbonyl glycine and 6-[(N-benzyloxycarbonyl)amino]hexanoic acid were synthesized according to the published procedure (15). R-(N-Benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA] was synthesized according to the literature method (16). (Dimethylamino)pyridine toluenesulfonate (DPTS) was prepared as described previously (17). Other regents were commercially available and used as received unless otherwise noted. All solvents were thoroughly dried and distilled before use. Measurements. 1H NMR spectra were recorded with a Bruker AXR-400 spectrometer operated at 400 MHz, CDCl3 was used as solvent, and tetramethylsilane was used as an internal standard. The average molecular weights and molecular weight distributions were measured on a Waters 410 gel permeation chromatography (GPC) equipped with three Ultrastyragel columns (104, 103, and 500 Å pore size) at room temperature. The eluent was THF, and polystyrene standards were used as calibrations. Synthesis of Poly(E-caprolactone) (PCL-OH) (18). Dried toluene (40 mL) and 10 mL of -CL solution in toluene (0.1 mol) were injected through a syringe into a flask which had been dried, purged with nitrogen, and vacuumized several times. Then a solution (0.11 g/mL, 9.27 mL) of Al(OiPr)3 in toluene was introduced at 0 °C to the reactor under nitrogen. The reaction mixture was incubated for 2 h at 0 °C and then diluted with 80 mL of toluene. Afterward, the solution was washed with 0.3 N HCl aqueous solution (3 × 20 mL) and water (3 × 30 mL). The organic phase was obtained and dried over MgSO4. The solution was concentrated to about 10 mL by a rotary evaporator and poured into 100 mL of hexane. The precipitated polymer was obtained by filtration and was redissolved in 10 mL of CHCl3, precipitated again in hexane. The purified product was dried under vacuum at room temperature, yielding 9 g of a white powder. Yield: 90%, Mw/Mn ) 1.33, Mn ) 4900. Synthesis of PCL-Cn-NH-Z and MPEO-Cn-NH-Z (16). (1) PCL-Cn-NH-Z. A 250 mL round-bottomed flask, fitted with a magnetic stir bar, was charged with 4 g of PCL-OH (1.67 mmol), 0.52 g of DCC (4.7 mmol), 0.29 g of DPTS (0.98 mmol), and 100 mL of CH2Cl2. To this mixture was added 0.53 g (2 mmol) of 6-[(N-benzyloxycarbonyl)amino]hexanoic acid or 0.42 g (2 mmol) of N-benzyloxycarbonyl glycine. The reaction mixture was stirred at room temperature for 48 h. After filtration, the solution was reduced in volume to 20 mL by a rotary evaporator, and the polymer was precipitated into cold methanol (400 mL), dissolved in chloroform (30 mL), reprecipitated out by cold methanol (200 mL), and dried under vacuum. PCL-C6-NH-Z: 65%; PCL-C2-NH-Z: 53%. (2) MPEO-Cn-NH-Z. These polymers were prepared from MPEO under the same conditions as in the case of PCL. But the purification procedure is different. The resulting reaction mixture was first precipitated into cold diethyl ether (400 mL), and the products obtained were dissolved in 40 mL of THF. After filtration, the solution was concentrated and precipitated into cold diethyl ether and dried in a vacuum oven. MPEO-C6-NH-Z: 79%; MPEO-C2-NH-Z: 85%. Synthesis of PCL-Cn-NH2 and MPEO-Cn-NH2. (1) PCL-Cn-NH2. A suspension of PCL-Cn-NH-Z (2.0 g) in

Scheme 1

EtOAc/MeOH (v/v, 2:1) (40 mL) and 1.0 g of 10% Pd supported on activated carbon was subjected to hydrogenation under a H2 blanket at room temperature for 24 h. After filtration over Celite, the solution was concentrated and precipitated into cold methanol. The obtained polymer was reprecipitated from cold methanol by dissolving in chloroform. PCL-C6-NH2: 70%, Mn ) 4800, Mw/ Mn ) 1.21; PCL-C2-NH2: 60%, Mn ) 4200, Mw/Mn ) 1.34. (2) MPEO-Cn-NH2. The polymers were prepared by using a similar procedure as for PCL-Cn-NH2, except that the precipitator was cold diethyl ether. MPEO-C6-NH2: 87%, Mn ) 2100, Mw/Mn ) 1.06; MPEO-C2-NH2: 80%, Mw/Mn ) 2100, Mn ) 1.10. Synthesis of Poly[ethylene oxide-β-(N-benzyloxycarbonyl-L-lysine)] [MPEO-β-P(lys(Z))] (4e). A 4.5 g aliquot of R-(N-benzyloxycarbonyl-L-lysine) N-carboxy anhydride (15 mmol) was dissolved in 45 mL of DMF. The resultant solution was added to a solution of MPEOC2-NH2 (2 g, 1 mmol) in DMF (70 mL). The reaction mixture was stirred under an inert atmosphere for 72 h at 40 °C and was precipitated into cold diethyl ether, followed by filtration and drying under reduced pressure to give 5.8 g of white powder. Yield: 89%, Mw/Mn ) 1.19, Mn ) 6000. Elemental analysis was conducted. Assume the average degree of polymerization is 15, Calcd for C303H457N31O92 (%): C, 60.61; H, 7.62; N, 7.23. Found: C, 60.51; H, 7.83; N, 7.28. RESULTS AND DISCUSSION

The amine-terminated MPEO and PCL were accomplished via a two-step process from hydroxyl-terminated MPEO and PCL as shown in Scheme 1. The hydroxylterminated PCL was prepared by the well-known coordination-insertion ring-opening polymerization of -caprolactone (-CL) in toluene using Al(OiPr)3 as an initiator (18). In our experiment, the reaction temperature was 0 °C, and the polymerization time was 2 h. 1H NMR spectrum of the resulting PCL is shown in Figure 1A. It can be seen that typical signals due to -CH2-OH, (CH3)2CHO-, and (CH3)2CHO- are observed at 3.62, 1.22-1.24, and 4.97-5.04 ppm, respectively, and their molar ratio was 2:6:1, confirming the R-hydroxyl ω-isopropyl structure. Based on the integral ratio of -OOC-CH2-CH2- at 2.27-2.36 ppm to (CH3)2CHO- at 1.22-1.24 ppm, the average molecular weight of the product was calculated to be 2400. The average molecular weight obtained by GPC measurements shown in Figure 2A is 4900. This value is larger than that derived from 1H NMR, which is normal when PS standards are used for GPC measurements (9, 20). The molecular weight distribution is 1.33. The condensation of hydroxyl-terminated PCL and MPEO with an excess of N-benzyloxycarbonyl amino acid was easily done at room temperature according to the literature approach (19). To confirm whether the reaction

Bioconjugate Chem., Vol. 13, No. 5, 2002 1161

Figure 3. 1H NMR spectra of MPEO. (A) MPEO-C2-NH-Z. (B) MPEO-C2-NH2.

Figure 1. 1H NMR spectra of (A) PCL-OH, (B) PCL-C6-NH-Z, and (C) PCL-C6-NH2.

Figure 2. GPC trace of PCL (A) hydroxyl-terminated and (B) amine-terminated.

is applicable for both R-amino acids and R,ω-amino acids, two different amino acids, N-benzyloxycarbonyl glycine and 6-[(N-benzyloxycarbonyl)amino]hexanoic acid, were tested. The reaction conditions were the same. The purification procedure was different for PCL and MPEO. In the case of PCL, purification was easily done by precipitation into cold methanol. Due to the good solubility of DPTS and DCC and the excess of amino acid in methanol, they were easily removed to give pure polymer. But in the case of MPEO-Cn-NH-Z, the solubility of DPTS in cold diethyl ether was not high enough, and the product may contain little DPTS if the final solution was precipitated out directly from diethyl ether. However, DPTS could be eliminated by filtrating the concentrated mixture of DPTS and MPEO-Cn-NH-Z in THF before precipitation into diethyl ether. The 1H NMR spectrum of PCL-C6-NH-Z is shown in Figure 1B. The emergence of the peaks at 5.10, 7.36 ppm assignable to the benzyloxycarbonyl protons and the disappearance of the peaks at 3.62 ppm belonging to the methylene group near the hydroxyl terminal indicate condensation is taking place. Figure 3 A is the 1H NMR spectrum of MPEO-C2-NH-Z. The triple peaks at 4.30-4.32 ppm are from the methylene groups adjacent to the newly formed ester bonds (-OCH2CH2OOC-). Again, the peaks belonging to protective groups appear at 5.1 and 7.30-7.36 ppm, respectively. Based on the intensity ratio of benzyl or methylene groups adjacent to the ester bond to unchangeable

terminal groups [(CH3)2CH- for PCL-C6-NH-Z, CH3O- for MPEO-C2-NH-Z], the efficiencies of the end-group functionalization are close to 100%. PCL-C2-NH-Z and MPEOC6-NH-Z gave similar results. The relatively lower yields of the resulting polymers were due to the solubility of polymers in methanol or diethyl ether. Catalytic hydrogenation is one of the most important methods to liberate the N-benzyloxycarbonyl-protected amino group. The catalyst is 10% Pd supported on activated carbon. The reaction condition is mild and is not sensitive to environmental factors such as water moieties. The reaction is known to proceed at the surface of Pd; therefore, once the stirring is gentle enough and H2 is sufficient, the reaction could proceed smoothly and completely. However, due to the adsorption of the polymer on Celite, the filtration over Celite to remove Pd/C decreases the yields. It can be seen from the 1H NMR spectrum of PCL-C6-NH2 as shown in Figure 2C that the signals at 5.10, 7.36 ppm disappear, while the signals at 3.21 ppm assignable to the methylene groups adjacent to amino groups shift to higher field, indicating complete deprotection. In the 1H NMR spectrum of MPEO-C2-NH2 (Figure 3B), the disappearance of the peaks belonging to the protective groups at 5.31, 7.30-7.36 ppm indicates that the benzyloxy carbonyl groups have been removed. At the same time, the signals assignable to -CH2CH2OOC- at 4.3 ppm remain, and the ratio of this peak to the methyl end group is 2:3, so the ester bond of the main chain does not change during the reaction. The structure of PCL-C6-NH2 was also confirmed by the 13C NMR spectrum as shown in Figure 4. The signal at 64.08 ppm is the characteristic peak assignable to the methylene close to the amino group, and the peaks due to (CH3)2CH-O- and -CH2-OCO- are overlapped with the signals of CDCl3. The GPC trace of PCL-C6-NH2 displays a single and sharp peak as shown in Figure 2B. Compared with the hydroxyl precursor, no obvious change of average molecular weight and molecular weight distributions is observed. It reveals that PCL was stable under the present conditions and little or no transesterification occurred. By increasing of the reaction time to 4 days, a decrease of molecular weight and an increase of molecular weight distributions were found, which might be caused by hydrolysis and transesterification. This phenomenon was reported by Langer et al. in other similar reactions (21),

1162 Bioconjugate Chem., Vol. 13, No. 5, 2002 Scheme 2

15, which confirmed that the initiation reactivity of MPEO-C2-NH2 was high. In conclusion, amine-terminated PCL and MPEO can be prepared conveniently from their hydroxyl-terminated precursors under mild conditions. This method may also be used for the preparation of other kinds of aminecontaining polymers from the hydroxyl precursors, and may even be used for R,ω-diamine-terminated polymers. Figure 4.

13C

NMR spectrum of PCL-C6-NH2.

ACKNOWLEDGMENT

This work was partially supported by the NSFC (Grant 20074002) and the Ministry of Education, China. LITERATURE CITED

Figure 5. GPC trace of MPEO-β-P[Lys(Z)].

Figure 6.

1H

NMR spectrum of MPEO-β-P[Lys(Z)].

while in the case of MPEO such a side reaction is less possible. To test the reactivity of the amine-terminated polymers prepared above, MPEO-C2-NH2 was used as a macroinitiator to initiate the ring-opening polymerization of a cyclic monomer of lysine, R-(N-benzyloxycarbonyl-Llysine) N-carboxy anhydride [lys(Z)-NCA] (Scheme 2). The targeted degree of polymerization of lys(Z)-NCA is designed to be 15. The GPC trace of the resulting copolymer (Figure 5) is a single peak, and the molecular weight distribution of the product is 1.19, showing that the reactivity of MPEO-C2-NH2 is high enough and the final product does not contain unreacted MPEO-C2-NH2. The 1H NMR spectrum of MPEO-β-P[lys(Z)] is shown in Figure 6. Besides the signals of the PEO block, new peaks at about 7.26, 1.20-1.90, 5.10, and 5.55 ppm corresponding to the N-protected polypeptide block appeared. From the integral ratio of CH3-O- (3.38 ppm) to -CH2-NH-Z (3.12 ppm), the average degree of polymerization (DP) of the cyclic monomer is calculated to be 16. Based on the elemental analysis of the final copolymer, the DP is

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