Biomacromolecules 2003, 4, 477-480
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Synthesis of Poly(L-lactide) End-Capped with Lactose Residue Tatsuro Ouchi,* Tomoyuki Uchida, Hidetoshi Arimura, and Yuichi Ohya* Faculty of Engineering & High Technology Research Center, Kansai University, Suita, Osaka 564-8680, Japan Received October 11, 2002; Revised Manuscript Received March 10, 2003
The synthesis of poly(L-lactide) (polyLA) end-capped with lactose residue was studied from the standpoint of development of a new bioabsorbable material. After the hydroxyl group of t-butoxycarbonyl(Boc)aminoethanol was converted to Boc-aminoethanol-OK by using potassium/naphthalene, L-lactide was polymerized in tetrahydrofuran using Boc-aminoethanol-OK as an initiator at room temperature to prepare polyLA-NHBoc. Subsequently, the removal of the Boc group in terminal Boc-aminoethanol residue was performed by treatment of formic acid to obtain the amino group end-capped polyLA (polyLA-NH2) as a reactive polyLA derivative. The coupling reactions of lactose with polyLA-NH2 were investigated by two methods; the synthetic method through reductive amination of lactose with polyLA-NH2 in the presence of sodium cyanoborohydride as a reducing agent did not give high degree of substitution of end-capped lactose residue per polyLA molecule, whereas the synthetic method through the ester interchange reaction of lactonolactone with polyLA-NH2 gave Lac-polyLA perfectly end-capped with lactose residue. Introduction Poly(L-lactide) (polyLA; PLA) is a biodegradable polyester having good biocompatibility. So, it has been utilized as a useful bioabsorbable material in the medical and pharmaceutical fields.1-9 However, the application scope of polyLA is limited because it is a polyester having no highly reactive group as a trigger of chemical reaction and the surface of polyLA matrix is very hydrophobic. To expand its application scope, the introduction of many kinds of hydrophilic units into polyLA has been tried. For example, the synthesis of the ABA-type of triblock copolymers composed of polyLA, poly(ethylene glycol) (PEG), and polyLA segments was reported.10-13 These copolymers were confirmed to form the microphase separation structure in which peptide drugs such as growth factor were found to be loaded into the hydrophilic PEG domains. We reported previously the synthesis of polyLA-grafted polysaccharides using polysaccharides as biodegradable hydrophilic backborn polymers through the trimethylsilyl protection, ring-opening polymerization of L-lactide (L-LA), and deprotection techniques.14-16 The polyLA-grafted polysaccharide was confirmed to show high biodegradability and to give a microphase separation structure.15 PolyLA having R-glucose as the monosaccharide at the end group was also synthesized using a D-glucose derivative protected with four benzyl groups having one hydroxyl group as an initiator to improve the wettability on polyLA film.17 However, many reaction steps through protection and deprotection were demanded to obtain these hybrid polymers of saccharide and polyLA. To solve these problems, we would like to propose the reaction procedure through the coupling reaction of lactose * To whom correspondence should be addressed. Phone: +81-6-63680814. Fax: +81-6-6339-4026. E-mail:
[email protected] (T.Ouchi);
[email protected] (Y.Ohya).
or a lactose derivative with polyLA having a reactive amino end group. Kataoka et al.18 already reported the synthesis of the diblock copolymer composed of polyLA and poly(ethylene glycol) segments having aldehyde as a reactive end group. Stassen et al.19 and Go¨pferich et al.20 reported previously the syntheses of the ω-aliphatic primary amine polyLA and amine-reactive diblock copolymer consisting of polyLA and poly(ethylene glycol)-amine segments, respectively. We also selected amino group as the reactive end group of polyLA. The direct coupling reaction of lactose or lactonolactone with polyLA having an amino end group (polyLA-NH2) was carried out to obtain the objective polyLA end-capped with lactose residue (Lac-polyLA). Experimental Section Materials. L-LA purchased from Boehringer Ingelheim (Milwaukee, WI) was recrystallized twice from ethyl acetate before using. 2-Aminoethanol purchased from Kanto Chemical (Tokyo, Japan) was used without further purification. Boc2O (Boc: t-butoxycarbonyl) and sodium cyanoborohydride were purchased from Peptide Institute, Inc. (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO), respectively. Naphthalene and other chemicals were purchased from Wako Pure Chemical Co. (Tokyo, Japan). Dry tetrahydrofuran (THF) purchased from Wako Pure Chemical Co. was used as a polymerization solvent without purification. Lactose was purchased from Kanto Chemical (Tokyo, Japan). The lactonolactone was prepared according to the method described in ref 21. Measurements. 1H NMR and 13C NMR spectra were recorded on a JEOL GSX-400 using tetramethylsilane (TMS) as the internal reference. Gel-permeation chromatography (GPC) analysis was carried out using a Tosoh GPC-8020 series system with a refractive index (RI) detector under the
10.1021/bm020110t CCC: $25.00 © 2003 American Chemical Society Published on Web 04/19/2003
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Table 1. Results of Synthesis of polyLA-NH2 anionic polymerizationa run
M/I
conv.c
1 2 3
50 100 200
98.2 98.0 98.5
yield (wt%) 80.4 85.5 92.2
deprotectionb
Mn
Mw/Mnd
DPLe
DIA
yield (wt%)
Mnd
Mw/Mnd
10 100 17 000 31 000
1.5 1.4 1.8
70 118 215
100 100 100
87.5 95.5 92.0
9500 15 000 30 000
1.3 1.5 1.3
d
a Polymerization was carried out for 30 min in THF at r.t. The initial concentration of L-LA was 2.5 mol/L. b Deprotection was carried out in the mixture of formic acid and CHCl3 (1/1, v/v) at r.t. for 9 h. c The conversion of L-LA to BocNH-PLA was estimated from the ratio of the integration value of 4.92-4.97 ppm assigned to the methyne proton signal of L-LA to that of 5.14-5.19 ppm assigned to the nonterminal methyne proton signal of polyLA chain in 1H NMR of reaction mixture. d Estimated by GPC. e Degree of polymerization of L-LA.
following condition: TSK Gel Multipore HXL-M×2 columns and THF eluent at the flow rate of 1 mL/min. The calibration curve for GPC analysis was obtained using polystyrene standards. The measurement of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF-MS) for the Lac-polyLA-2B (Mn ) 10 100; Mw/Mn ) 1.2) sample was performed using a Finnigan MAT Vision 2000 time-of-flight mass spectrometer. This instrument is equipped with a N2 laser (337 nm) and a reflector. It was operated at an accelerating potential of 25 kV in reflector mode. The matrix, 2,5-dihydroxybenzoic acid (DHB), was also dissolved in THF. The final solution was deposited onto the sample target and allowed to dry in air at room temperature. Internal standards (DHB and insulin, bovine pancreas) were used to calibrate the mass scale. Synthesis of Boc-aminoethanol.22 2-Aminoethanol (0.61 g, 10 mmol) was dissolved in chloroform (CHCl3; 20 mL) in a flask equipped with a magnetic stirrer, and Boc2O (2.4 g, 11 mmol, 1.1eq) in CHCl3 (20 mL) was added. After stirring for 30 min, the concentrated solution was diluted with 5% potassium hydrogensulfate aq. soln. and extracted with ethyl acetate and dried over sodium sulfate. The purification of the compound was carried out by column chromatography on silica gel using CHCl3 and methanol as eluent. The purity of Boc-aminoethanol obtained as a colorless syrup was found to be over 99.8% by HPLC analysis (column: ODS-120T). Yield: 82.0%. 1H NMR (CDCl3): δ (ppm) ) 1.43 (s, 9H, C(CH3)3), 3.26 (t, 2H, NCH2CH2OH), 3.68 (t, 2H, NCH2CH2OH). 13C NMR (CDCl3): δ (ppm) ) 28.4 (3C, C(CH3)3), 43.5 (1C, CH2OH), 67.4 (1C, CH2NH), 79.9 (1C, C(CH3)3), 256.8 (1C, COO). Polymerization of L-LA with Boc-aminoethanol. The following procedures were basically carried out in a dry glove box under a N2 atmosphere. Boc-aminoethanol was converted to the corresponding alkoxide (Boc-aminoethanol-OK) using potassium/naphthalene, and then L-LA was polymerized using Boc-aminoethanol-OK as an initiator to obtain polyLANHBoc. Potassium/naphthalene was prepared by adding potassium to the solution of naphthalene (128 mg, 1.0 mmol) dissolved in dry THF (2 mL). One mL of this solution was added to Boc-aminoethanol (40 mg, 0.25 mmol) dissolved in dry THF (1 mL). After stirring for 10 min, 5-20 mL of L-LA in THF (3.6 M) was added (M/I ) 50-200). When the reaction proceeded at room temperature for 30 min, the polymerization was terminated by addition of acetic acid to the reaction mixture. The reaction mixture was poured into a large amount of diethyl ether to precipitate the polymerization product. The resulting product as a white solid was further reprecipitated with CHCl3/diethyl ether and dried
Scheme 1. Synthetic Route of polyLA-NH2
under vacuum overnight at room temperature. Characterization of the polymerization product was performed by 1H NMR and GPC measurements. The results of Mn, Mw/Mn, and yield are shown in Table 1. 1H NMR (CDCl3): δ (ppm) ) 1.44 (s, 9H, CHCH3 of Boc group), 1.62 (d, 3H, CHCH3 of polyLA), 3.39 (br, 2H, NCH2CH2O), 4.20 (br, 2H, NCH2CH2O), 4.36 (q, 1H, terminal-CHCH3), 5.17 (q, 1H, CHCH3 of polyLA). 13C NMR (CDCl3): δ (ppm) ) 16.7 (1C, CHCH3), 69.3 (1C, CHCH3), 169.4 (1C, CO). Deprotection. The removal of the Boc group from the obtained polyLA-NHBoc was performed by treatment in the mixing solvent of formic acid (20 mL) and CHCl3 (20 mL). After 9 h of treatment at room temperature, the solution was poured into a large amount of diethyl ether to obtain the precipitate. The precipitate was dried under vacuum at room temperature, and then the product was treated in the mixing solvent of triethylamine (TEA; 20 mL) and CHCl3 (20 mL) to deprotonate at room temperature for 8 h. The purification of polyLA-NH2 was carried out by the method similar to that of polyLA-NHBoc. The removal of the Boc group from polyLA-NHBoc was confirmed by the perfect disappearance of the methyl proton signal at 1.44 ppm in the 1H NMR spectrum. Characterization of the product was performed by 1H NMR and GPC measurements. The results of M , M / n w Mn and yield are shown in Table 1. 1H NMR (CDCl3): δ (ppm) ) 1.62 (d, 3H, CHCH3), 3.42 (t, 2H, NCH2CH2O), 3.71 (t, 2H, NCH2CH2O), 4.36 (q, 1H, terminal-CHCH3), 5.17 (q, 1H, CHCH3). 13C NMR (CDCl3): δ (ppm) ) 16.7 (1C, CHCH3), 69.3 (1C, CHCH3), 169.4 (1C, CO). Coupling Reaction of Lactose with polyLA-NH2 by Reductive Amination (Method 1 in Scheme 2).23-25 This reaction was carried out in a dry condition. Lac-polyLA-1 was synthesized in dimethyl sulfoxide (DMSO) by reductive amination of lactose with polyLA-NH2 in the presence of sodium cyanoborohydride at 60 °C for 60 h. Lac-polyLA-1
Biomacromolecules, Vol. 4, No. 3, 2003 479
Communications Scheme 2. Synthesis of Lac-polyLAs by Two Methods
Table 2. Results of Coupling Reaction of Disaccharide with polyLA-NH2 run
disaccharide
Lac-polyLA-1 lactosea Lac-polyLA-2A lactonolactoneb Lac-polyLA-2B lactonolactonec
recovery (wt%)
Mnd
77.4 70.5 50.6
11 000 9900 10 100
1H, terminal-CHCH3), 4.29 (d, 1H, anomer), 5.20 (q, 1H, CHCH3).
DSL Mw/Mnd (mol%) 1.2 1.2 1.2
34 87 100
a Reductive amination of lactose with polyLA-NH (Mn)9500, Mw/ 2 Mn)1.3) was carried out at 20 molar ratio of lactose to polyLA-NH2 in b DMSO at 60 °C for 60h. Condensation of lactonolactone with polyLANH2 (Mn)9500, Mw/Mn)1.3) was carried out at 4 molar ratio of lactonolactone to polyLA-NH2 in DMF at 80 °C for 24h. c Condensation of lactonolactone with polyLA-NH2 (Mn)9500, Mw/Mn)1.3) was carried out at 8 molar ratio of lactonolactone to polyLA-NH2 in DMF at 80 °C for 24 h. d Estimated by GPC.
purified by reprecipitation with CHCl3/(diethyl ether: methanol)1:1) was dried overnight under vacuum at room temperature. It was confirmed by TLC using methanol as an eluent that unreactive lactose was not contained in the obtained product. The degree of substitution of the endcapped lactose residue per polyLA molecule (DSL) for the Lac-polyLA-1 product was calculated from the ratio of the integration value of the peak at δ 4.20 assigned to the terminal methyne proton signal of the polyLA chain to that of δ 4.29 assigned to the anomer proton signal of the galactose unit in the 1H NMR spectrum. The results of Mn, Mw/Mn, DSL, and recovery of the products are shown in Table 2. 1H NMR (DMSO-d6 addition to D2O, at 60 °C): δ (ppm) ) 1.47 (d, 3H, CHCH3), 3.06-3.67 (m, 18H, disaccharide and methylene of aminoethanol), 4.20 (q, 1H, terminal-CHCH3), 4.29 (d, 1H, anomer), 5.20 (q, 1H, CHCH3). Coupling Reaction of Lactonolactone with polyLANH2 (Method 2 in Scheme 2). The coupling reaction of lactonolactone with polyLA-NH2 was carried out in N,Ndimethylformamide (DMF) at 80 °C for 24 h to obtain LacpolyLA-2. The purification and characterization were carried out by the same methods described in the method 1. The results of Mn, Mw/Mn, DSL, and recovery of the products are shown in Table 2. 1H NMR (DMSO-d6 addition to D2O, at 60 °C): δ (ppm) ) 1.47 (d, 3H, CHCH3), 3.06-3.67 (m, 18H, disaccharide and methylene of aminoethanol), 4.20 (q,
Results and Discussion The amino group end-capped polyLA could be prepared through the synthetic route shown in Scheme 1. The hydroxyl group of Boc-aminoethanol was converted to Boc-aminoethanol-OK by using potassium/naphthalene. The anionic ring-opening polymerization of L-LA was carried out at room temperature by using Boc-aminoethanol-OK as an initiator. The results of the polymerization of L-LA are summarized in Table 1. The degree of polymerization of polyLA-NHBoc was found to be controlled by changing the feed molar ratio of L-LA to Boc-aminoethanol-OK (M/I) (Table 1). The degree of introduction of Boc-aminoethanol residue per polyLA molecule (DIA) was calculated from the ratio of integration value of 4.36 ppm assigned to terminal methyne proton signal of polyLA chain to that of 3.39 ppm assigned to methylene proton signals of the end-capped Boc-aminoehanol residue in the 1H NMR spectrum. As a result, the Boc-aminoethanol residue was quantitatively introduced at polyLA-terminal. The removal of the Boc group of the terminal Bocaminoethanol residue from the obtained polyLA-NHBoc was performed by treatment in the mixed solvent of formic acid and CHCl3 at room temperature for 9 h and by subsequent deprotonation of -NH3+ with TEA in CHCl3 at room temperature for 8 h. The perfect deprotection of the Boc group from polyLA-NHBoc was confirmed by the disappearance of the methyl peak; we succeeded in preparing the perfectly deprotected NH2 end-capped polyLA, polyLA-NH2, having various molecular weights (Table 1). Moreover, the decrease of the molecular weight of the polyLA segment for the obtained polyLA-NH2 was hardly found through the deprotection (Table 1). It was proved from the ratio of integration value of 4.36 ppm assigned to the terminal methyne proton signal of the polyLA chain to the integration value of 3.42 ppm assigned to methylene proton signals of the end capped aminoethanol residue in the 1H NMR spectrum that the aminoethanol group was quantitatively introduced at polyLA-terminal. The obtained polyLA-NH2
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indicated the same solubility as homopolyLA; polyLA-NH2 was dissolved in CHCl3, THF, and DMF and not in water, methanol, and diethyl ether. The coupling reaction of lactose with polyLA-NH2 was performed by two methods (Scheme 2). Method 1 was the synthetic method through reductive amination of the aldehyde group of lactose with the amino group of polyLA-NH2. The reaction was performed with the feed ratio of lactose to polyLA-NH2 as 20 molar equiv in the presence of sodium cyanoborohydride in DMSO at 60 °C for 60 h to obtain LacpolyLA-1. Method 2 was the synthetic method through the ester interchange reaction of lactonolactone with polyLANH2. The reaction was carried out with the feed ratio of lactonolactone to polyLA-NH2 as 4-8 molar equiv in DMF at 80 °C for 24 h to obtain Lac-polyLA-2. It was confirmed by TLC that unreactive lactose or lactonolactone was not contained in the reprecipitated products. The results of the coupling reaction of disaccharide with polyLA-NH2 are shown in Table 2. We succeeded in preparing Lac-polyLA perfectly end-capped with the lactose residue at 8 molar feed ratio of lactonolactone to polyLA-NH2 by Method 2. On the contrary, despite an investigation of the reaction condition, Lac-polyLA with a DSL of 100 mol % could not be obtained by Method 1. The low existence probability of aldhexose of reducing end in the equilibrium state could be pointed out as a reason of low DSL of Lac-polyLA-1. The lactose residue perfectly end-capped chemical structure for Lac-polyLA-2B was reconfirmed by MALDI-TOF-MS measurement. The MALDI-TOF-MS spectrum of Lac-polyLA-2B detected shows the following series: 6565, 6710, 6723, 6867, 7012, 7155, 7300, etc. These peak values agreed very closely with the calculated values from [Lac-(LA)n, Na+]. These results meant that the Lac-polyLA-2B sample had the lactose residue perfectly end-capped polyLA structure. The polyLA-NH2 obtained above is a very interesting reactive polyLA derivative to afford easily the polyLA based hybrid materials. So, many kinds of new hybrid materials of polyLA can be expected to be created from polyLA-NH2. Acknowledgment. This research was financially supported by a Grant-in-Aid for Scientific Research (B)(13558210) from the Japan Society for the Promotion of Science.
Communications
Supporting Information Available. Figure showing 1H NMR spectra of polyLA-NHBoc(CDCl3), polyLA-NH2(CDCl3), Lac-polyLA-1(DMSO-d6 and D2O), Lac-polyLA2B(DMSO-d6 and D2O). This material is free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Kulkarni, K. R.; Moore, G. E.; Hegyeli, F. A.; Leonard, F. J. Biomed. Mater. Res. 1971, 5, 169. (2) Wood, A. D. Int. J. Pharm. 1980, 7, 1. (3) Eling, S.; Gogolewski, B.; Pennings, J. A. Polymer 1982, 23, 1587. (4) Heller, J. CRC Crrit. ReV. Ther. Drug Carrier Syst. 1985, 1, 39. (5) Chabot, F.; Vert, M.; Chapelle, S.; Granger, P. Polymer 1983, 24, 53. (6) Jeoung, Y. S.; Kim, W. S. Arch. Pharm. Res. 1986, 9, 63. (7) Holland, J. S.; Tighe, J. B.; Gould, L. P. J. Controlled Release 1986, 4, 155. (8) Kricheldorf, R. H.; Kreiser-Samders, I. Macromol. Symp. 1996, 103, 85. (9) Langer, R. Acc. Chem. Res. 2000, 33, 94. (10) Kimura, Y.; Matsuzaki, Y.; Yamane, H.; Kitao, T. Polymer 1989, 30, 1342. (11) Hu, D. S. G.; Liu, H. J.; Pan, I. L. J. Appl. Polym. Sci. 1993, 50, 1391. (12) Ronneberger, B.; Kao, W. J.; Anderson, J. M.; Kissel, T. J. Biomed. Mater. Res. 1996, 30, 31. (13) Kissel, T.; Li, Y. X.; Volland, C.; Gorich, S.; Koneberg, R. J. Controlled Release 1996, 39, 315. (14) Ohya, Y.; Maruhashi, S.; Ouchi, T. Macromolecules 1998, 31, 4662. (15) Ohya, Y.; Maruhashi, S.; Ouchi, T. Macromol. Chem. Phys. 1998, 199, 2017. (16) Ydens, I.; Rutot, D.; Degee, P.; Six, J.; Dellacherie, E.; Dubois, P. Macromolecules 2000, 33, 6713. (17) Ouchi, T.; Uchida, T.; Ohya, Y. Macromol. Biosci. 2001, 1, 371. (18) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Biomacromolecules 2000, 1, 39. (19) Stassen, S.; Archambeau, S.; Dobois, Ph.; Jerome, R.; Teyssie, Ph. J. Polym. Sci. Part A: Polym. Chem. 1994, 32, 2443. (20) Tessmar, J. K.; Mikos, A. G.; Go¨pferich, A. Biomacromolecules 2002, 3, 194. (21) Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym. J. 1985, 17, 567. (22) Hunter, C.; Jackson, R.; Rami, H. J. Chem. Soc., Perkin Trans. 1 2000, 219. (23) Clinton, F. L. Synthesis 1975, 135. (24) Ferdous, A.; Akaike, T.; Maruyama, A. Biomacromolecules 2000, 1, 186. (25) Branzat, M.; Perez, E.; Rico-Lattes, I.; Prome, D.; Prome, J. C.; Lattes, A. Langmuir 1999, 15 (5), 6163.
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