Biomacromolecules 2010, 11, 1089–1093
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Lipase-Catalyzed Synthesis of Poly(amine-co-esters) via Copolymerization of Diester with Amino-Substituted Diol Zhaozhong Jiang* Biomedical Engineering Department, Yale University, 55 Prospect Street, New Haven, Connecticut 06511 Received January 18, 2010; Revised Manuscript Received February 16, 2010
Candida antarctica lipase B (CALB) was found to be an efficient catalyst for copolymerization of diesters with amino-substituted diols to form poly(amine-co-esters) in one step. The copolymerization reactions were carried out at 50-100 °C in two stages: first stage oligomerization under 1 atm pressure of nitrogen followed by second stage polymerization under 1-2 mmHg vacuum. The formed copolymers possessed molecular weight (Mw) up to 59000 and typical polydispersity (Mw/Mn) between 1.5 and 2.3. The enzymatic reaction appears to be quite general and accommodates a large number of comonomer substrates with various chain length and substituents. Thus, C4-C12 diesters (i.e., from succinate to dodecanedioate) and diethanolamine comonomers with either an alkyl (methyl, ethyl, n-butyl, t-butyl) or an aryl (phenyl) substituent on nitrogen were successfully incorporated into the poly(amine-co-ester) chains. Biodegradable polyesters bearing tertiary amino groups have been reported to be efficient carriers for gene delivery. The high tolerance of the lipase toward tertiary amino functional groups as described in this paper provides new routes for synthesizing poly(amine-co-esters) with tailored structures for specific biomedical applications.
Introduction Various types of polymeric materials containing amine functional groups have been used to serve as nonviral carriers for DNA (or gene) delivery to living cells.1 These polymers are capable of condensing plasmid DNA via electrostatic interactions to form nanosized polyelectrolyte complexes (or polyplexes), protecting DNA against extracellular nuclease degradation, and facilitating transportation of DNA into cell compartments through cellular barriers.2 Examples of such polymers include poly(ethyleneimine), poly(4-hydroxy-L-proline ester) (PHP), poly[R-(4-aminobutyl)-L-glycolic acid] (PAGA), poly(β-amino esters) (PBAE), poly(L-lysine), chitosan, poly(dimethylaminoethyl methacrylate), and poly(trimethylaminoethylmethacrylate).3 Among these materials, polyesters bearing tertiary amino substituents are particularly promising due to their biodegradability (thus avoiding accumulation of the polymers in the body after repeated administration), low cytotoxicity, and outstanding transfection efficacy.4 However, few efficient synthetic methods are currently available for preparation of amino-containing polyesters primarily because metal catalysts required for conventional polyester synthesis are often sensitive to and deactivated by amino groups. Synthesis of PBAEs was achieved via conjugate addition between diacrylate and amine comonomers.5,6 No catalysts are required for the addition polymerization reactions. On the other hand, low molecular weight PHP (Mw up to 9000 Da)7,8 and PAGA (∼3300 Da)9,10 were prepared in multiple steps, involving protection and deprotection of the amino substituents. Recently, synthesis of poly(N-methyldiethyleneamine sebacate) was reported via polycondensation reaction between sebacoyl chloride and N-methyldiethanolamine.11 Triethylamine in large excess was used to remove hydrochloric acid byproduct of the reaction. However, diacyl halides are expensive and unstable chemicals (e.g., * To whom correspondence should be addressed. Phone: 203-432-7638. Fax: 203-432-0030. E-mail:
[email protected].
sensitive to moisture), which render them undesirable to serve as comonomers for polyester synthesis. In the past two decades, enzymes (e.g., lipases) have been extensively evaluated as environmentally benign, alternative catalysts for polyester preparation.12,13 Various polyesters were successfully synthesized via enzymatic polymerization reactions including condensation copolymerization of dicarboxylic acids with diols,14 transesterification reaction of diesters with diols,14,15 polymerization of hydroxy acids,14 ring-opening polymerization of lactones,16,17 and combined ring-opening and condensation copolymerization of lactones with diesters and diols.18–21 Additionally, syntheses of aliphatic polycarbonates22–24 and poly(carbonate-co-esters)25,26 have also been achieved. Lipases are known to be highly tolerant of functional organic moieties (e.g., hydroxyl, vinyl, epoxy) and are ideally suited for synthesis of functional polyesters.12,13 Most recently, polyamides were prepared via copolymerization between diester and diamine comonomers.27 Herein, I report for the first time lipasecatalyzed, one-step synthesis of poly(amine-co-esters) via copolymerization of diesters with amino-substituted diols. The polymer molecular weights were measured by gel permeation chromatography (GPC) and the polymer structures were characterized by 1H and 13C NMR spectroscopy.
Experimental Section Materials. Diethyl succinate (99%), diethyl adipate (99%), diethyl suberate (97%), diethyl sebacate (98%), diethyl dodecanedioate (98%), N-methyldiethanolamine (99+%), N-ethyldiethanolamine (98%), N-nbutyldiethanolamine (98+%), N-tert-butyldiethanolamine (97%), Nphenyldiethanolamine (97%), and diphenyl ether (99%) were purchased from Aldrich Chemical Co. and were used as received. Immobilized Candida antarctica lipase B (CALB) supported on acrylic resin or Novozym 435, chloroform (HPLC grade), dichloromethane (99+%), hexane (97+%), and chloroform-d were also obtained from Aldrich Chemical Co. The lipase catalyst was dried at 50 °C under 2.0 mmHg for 20 h prior to use. Instrumental Methods. 1H and 13C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer. The number of scans used
10.1021/bm1000586 2010 American Chemical Society Published on Web 03/05/2010
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for acquiring 1H and 13C NMR spectra was 32-48 and 2000-2400, correspondingly. The chemical shifts reported were referenced to internal tetramethylsilane (0.00 ppm) or to the solvent resonance at the appropriate frequency. The number and weight average molecular weights (Mn and Mw, respectively) of polymers were measured by gel permeation chromatography (GPC) using a Waters HPLC system equipped with a model 1515 isocratic pump, a 717 plus autosampler, and a 2414 refractive index (RI) detector with Waters Styragel columns HT6E and HT2 in series. Empower II GPC software was used for running the GPC instrument and for calculations. Both the Styragel columns and the RI detector were heated and maintained at 40 °C temperature during sample analysis. Chloroform was used as the eluent at a flow rate of 1.0 mL/min. Sample concentrations of 2 mg/mL and injection volumes of 100 µL were used. Polymer molecular weights were determined based on a conventional calibration curve generated by narrow polydispersity polystyrene standards from Aldrich Chemical Co. General Procedure for CALB-Catalyzed Copolymerization of Diester with Amino-Substituted Diol. The polycondensation reactions were performed in diphenyl ether solution using a parallel synthesizer connected to a vacuum line with the vacuum ((0.2 mmHg) controlled by a digital vacuum regulator. In a typical experiment, reaction mixtures containing equal moles of diester and amino-substituted diol monomers, Novozym 435 (10 wt % vs total monomer), and diphenyl ether solvent (200 wt % vs total monomer) were prepared. The copolymerization reactions were carried out in two stages: first stage oligomerization followed by second stage polymerization. During the first stage reaction, the reaction mixtures were stirred at 50-100 °C under 1 atm pressure of nitrogen for 24 h. Thereafter, the reaction pressure was reduced to 1-2 mmHg and the reactions were continued for an additional 70-80 h. The same temperature was used for both oligomerization and polymerization steps. At the end of the reactions, the formed polymers were dissolved in HPLC-grade chloroform and filtered to remove the enzyme catalyst. Polymer products were not fractionated by precipitation prior to analysis of molecular weight and structure. The filtrates containing whole products were analyzed by GPC using polystyrene standards to measure polymer molecular weights. To determine polymer structures, the product mixtures were dissolved in chloroform-d. The resultant solutions were filtered to remove catalyst particles and then analyzed by 1H and 13C NMR spectroscopy. For a few selected polycondensation reactions, polymer chain growth versus reaction time was monitored. Thus, aliquots were withdrawn at various time intervals during the second stage polymerization and the polymer molecular weights were analyzed by GPC according to the method described above. Poly(N-methyldiethyleneamine succinate) (PMSN): 1H NMR (CDCl3; ppm) 2.33 (s, 3H), 2.63 (s, 4H), 2.69 (t, 4H), 4.18 (t, 4H); 13C NMR (CDCl3; ppm) 28.97, 42.79, 55.82, 62.39, 172.14. Poly(N-methyldiethyleneamine adipate) (PMAP): 1H NMR (CDCl3; ppm) 1.65 (br, 4H), 2.33 (br, 7H), 2.68 (t, 4H), 4.15 (t, 4H); 13C NMR (CDCl3; ppm) 24.29, 33.78, 42.85, 55.92, 62.03, 173.12. Poly(N-methyldiethyleneamine suberate) (PMSR): 1H NMR (CDCl3; ppm) 1.32 (br, 4H), 1.61 (quintet, 4H), 2.29 (t, 4H), 2.32 (s, 3H), 2.67 (t, 4H), 4.15 (t, 4H); 13C NMR (CDCl3; ppm) 24.67, 28.73, 34.08, 42.85, 55.92, 61.95, 173.45. Poly(N-methyldiethyleneamine sebacate) (PMSC): 1H NMR (CDCl3; ppm) 1.29 (br, 8H), 1.61 (quintet, 4H), 2.30 (t, 4H), 2.34 (s, 3H), 2.69 (t, 4H), 4.16 (t, 4H); 13C NMR (CDCl3; ppm) 24.85, 29.04, 29.07, 34.17, 42.85, 55.92, 61.93, 173.57. Poly(N-methyldiethyleneamine dodecanedioate) (PMDO): 1H NMR (CDCl3; ppm) 1.28 (br, 12H), 1.61 (quintet, 4H), 2.30 (t, 4H), 2.33 (s, 3H), 2.68 (t, 4H), 4.15 (t, 4H); 13C NMR (CDCl3; ppm) 24.90, 29.12, 29.24, 29.39, 34.22, 42.86, 55.94, 61.93, 173.65. Poly(N-ethyldiethyleneamine sebacate) (PESC): 1H NMR (CDCl3; ppm) 1.01 (t, 3H), 1.29 (br, 8H), 1.60 (quintet, 4H), 2.28 (t, 4H), 2.59 (quartet, 2H), 2.73 (t, 4H), 4.12 (t, 4H); 13C NMR (CDCl3; ppm) 12.13, 24.85, 29.04, 29.08, 34.17, 48.65, 52.28, 62.40, 173.52.
Jiang Scheme 1. Two-Stage Process for Copolymerization of Diesters with Amino-Substituted Diols
Poly(N-n-butyldiethyleneamine sebacate) (PBnSC): 1H NMR (CDCl3; ppm) 0.90 (t, 3H), 1.29 (br, 10H), 1.40 (quintet, 2H), 1.60 (quintet, 4H), 2.27 (t, 4H), 2.50 (t, 2H), 2.72 (t, 4H), 4.11 (t, 4H); 13C NMR (CDCl3; ppm) 14.05, 20.34, 24.87, 29.07, 29.09, 29.59, 34.18, 52.84, 54.78, 62.41, 173.50. Poly(N-tert-butyldiethyleneamine sebacate) (PBtSC): 1H NMR (CDCl3; ppm) 1.04 (br, 9H), 1.29 (br, 8H), 1.61 (quintet, 4H), 2.27 (t, 4H), 2.74 (t, 4H), 4.05 (t, 4H); 13C NMR (CDCl3; ppm) 24.86, 27.05, 29.06, 29.08, 34.14, 49.55, 54.71, 64.72, 173.51. Poly(N-phenyldiethyleneamine sebacate) (PPSC): 1H NMR (CDCl3; ppm) 1.25 (br, 8H), 1.57 (br, 4H), 2.25 (t, 4H), 3.55 (t, 4H), 4.20 (t, 4H), 6.68 (t, 1H), 6.74 (d, 2H), 7.19 (t, 2H); 13C NMR (CDCl3; ppm) 24.75, 28.99, 29.03, 34.02, 49.49, 61.12, 111.99, 116.90, 129.38, 147.19, 173.46. Isolation and Purification of Poly(amine-co-esters). To purify the polymers formed according to the procedures described above, hexane was added to the product mixtures to cause precipitation of the poly(amine-co-esters). The precipitated soft materials or viscous oils were washed with hexane three times to extract and remove diphenyl ether solvent from the polymeric materials. Subsequently, the poly(amineco-esters) were dissolved in dichloromethane followed by filtration to remove the catalyst particles. The resultant filtrates were concentrated under vacuum and then dried at 40 °C under high vacuum (
