Glutathione-Mediated Biodegradable Polyurethanes Derived from l

Biomacromolecules 2010, 11, 269–276

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Glutathione-Mediated Biodegradable Polyurethanes Derived from L-Arabinitol M. Violante de Paz, Francisca Zamora, Beleń Begines, Cristina Ferris, and Juan A. Galbis* Dpto. Quı´mica Orgańica y Farmaceútica, Facultad de Farmacia, Universidad de Sevilla, 41012-Sevilla, Spain Received October 1, 2009; Revised Manuscript Received November 21, 2009

The synthesis, characterization, and some properties of new glutathione-mediated biodegradable sugar-based copolyurethanes are described. These copolyurethanes were obtained by polyaddition reaction of mixtures of 2,2′-dithiodiethanol (DiT) and 2,3,4-tri-O-benzyl-L-arabinitol (ArBn) or 2,3,4-tri-O-methyl-L-arabinitol (ArMe) to 1,6-hexamethylene diisocyanate (HMDI). The copolymer compositions were studied by elemental microanalyses and 1H NMR, revealing that the content of the copolymer units is in all cases very similar to that of their corresponding feed. The PU(DiT-HMDI) homopolymer exhibited a high crystallinity, but the introduction of the arabinitol-based diols led to a reduction in the crystallinity of the copolymers. In their TG curves, the copolymers exhibited a mixed trend of the related homopolymers, and all of them were thermally stable, with degradation temperatures above 220 °C. The degradation properties of the macromolecules under physiological conditions in the presence of glutathione were tested. All the copolyurethanes proved to be biodegradable under the experimental conditions (pH ) 7.02 and 37 °C). The degradation pattern of the copolymers depended not only on the dithiodiethanol (DiT) reactive units ratio in the polymer backbone, but also on the crystallinity of the macromolecule.

Introduction The preparation and applications of polyurethanes have been extensively studied for the last few decades, and they have been the subject of numerous patents, papers, and books. Most polyurethanes are used as commodity materials and have industrial applications;1,2 some of them are biodegradable and biocompatible materials.3-6 Therefore, the use of polyurethanes in medical applications is being widely investigated due to their low toxicity, potential biodegradability, biocompatibility,7 and versatile structures, which make them suitable as part of drug administration systems,8,9 as dermatological dressings,10,11 as hemocompatible materials for catheters,12 and as filters in biomedical instrumentation and devices.13-15 The main goal of numerous papers and reviews in the bibliography has been to report the different degradation mechanisms of polyurethanes (mainly hydrolytic, enzymatic, and oxidative pathways).16-20 In our research group, we are interested in the preparation of new polyurethanes in order to make them biocompatible and more biodegradable.21,22 In previous studies, we verified that the hydrolytic degradation of L-arabinitol and xylitol-based polyurethanes did not result in great reductions in their average molecular weight (Mw) unless drastic experimental conditions were used (reductions of 60% in Mw were achieved at pH 10.0 and 80 °C, after 39 days).23 Searching for different approaches to enhance the degradability of polyurethanes in biological systems, we thought of disulfide bonds, as they are the bridging structure most commonly encountered in biological systems, and can be cleaved by the action of the natural tripeptide glutathione (γ-glutamylcysteinyl-glycine; GSH). It is the most abundant low-molecularweight biological thiol, and one of nature’s premier antioxidants and free-radical scavengers.24 Glutathione is found in a wide variety of plant and animal tissues, where it also serves as an oxygen carrier in respiration and as a coenzyme in certain * To whom correspondence should be addressed. E-mail: [email protected].

metabolic processes. Glutathione exists in reduced (GSH) and oxidized (glutathione disulfide, GSSG) states, where the GSH/ GSSG system is the major redox couple in animal cells.25 Disulfide bonds are amply stable in the circulation and in the extracellular media, but they are prone to rapid cleavage under a reductive environment through the fast and readily reversible thiol-disulfide exchange reactions.26,27 This quick-response chemical degradation contrasts sharply with common hydrolytically degradable pathways for polymers such as aliphatic polyesters and polycarbonates, whose ester and carbonate bonds usually exhibit gradual degradation kinetics inside the body.28-30 Reduction-sensitive biodegradable polymers and conjugates have emerged as a fascinating class of biomedical materials that can be elegantly applied for drug delivery systems, as shown in the interesting review of Zhong et al.31 Such polymers incorporate disulfide linkage(s) that can be reduced by GSH in the body. This remarkable feature renders them distinct from their hydrolytically degradable counterparts and they are highly promising functional biomaterials that have enormous potential in formulating sophisticated drug and gene delivery systems. In this study, we propose the introduction of disulfide linkage into the polymer backbone of novel reduction-sensitive biodegradable sugar-based polyurethanes. Thus, we have designed, synthesized, and characterized a group of novel copolyurethanes (Scheme 1) derived from two diols and 1,6-hexamethylene diisocyanate (HMDI). This diisocyanate has proven to be suitable for the synthesis of polyurethanes with low toxicity for humans and, hence, for future uses as biomedical materials. The main diol monomer was a carbohydrate-based molecule, chosen between 2,3,4-tri-O-benzyl-L-arabinitol (ArBn) and 2,3,4-tri-O-methyl-L-arabinitol (ArMe). The other diol monomer, 2,2′-dithiodiethanol (DiT), was introduced as the carrier of the reactive segments for future degradations. It possesses a disulfide bond in its chemical structure and potentially could be reduced by the tripeptide glutathione under physiological conditions,

10.1021/bm9011216  2010 American Chemical Society Published on Web 12/02/2009

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Scheme 1. Synthesis of Copolyurethanes

Scheme 2. Synthesis of the Model Homopolyurethane PU(DiT-HMDI)

leading to the degradation of the polymer chain. The homopolymer PU(DiT-HMDI) was also prepared as a model compound (Scheme 2).

Experimental Section General Methods. Common reagents and solvents were purchased from Aldrich Chemical Co. and used as received. Solvents were dried and purified, when necessary, by appropriate standard procedures. 2,3,4Tri-O-benzyl-L-arabinitol (ArBn) and 2,3,4-tri-O-methyl-L-arabinitol (ArMe) were prepared from naturally occurring L-arabinose following known methods.21,37 Optical rotations were measured in a Perkin-Elmer 341 polarimeter 20 ( 0.5 °C (1 dm cell). Elemental analyses were determined in the Microanalyses Laboratories of the CITIUS Service in the Universidad de Sevilla. IR spectra (films or KBr discs) were recorded with a JASCO FT/IR-410 spectrometer. NMR spectra were recorded at 300 K on either a Bruker Advance AV-500 or a Bruker AMX-500. Chemical shifts (δ) are reported as parts per million downfield from Me4Si. Mass spectra were obtained using a Kratos MS80RFA instrument. Intrinsic viscosity measurements were carried out in dichloroacetic acid with a CannonUbbelohde 100/L30 semimicroviscosimeter at 25.0 ( 0.1 °C. Gel permeation chromatography (GPC) analyses were performed using a Waters apparatus equipped with a Waters 2414 refractive-index detector and two Styragel HR columns (7.8 × 300 mm) linked in series, thermostatted at 60 °C, using N-methylpyrrolidone (NMP) as the mobile phase, at a flow rate of 0.5 mL min-1. Molecular weights were estimated against polystyrene standards. The thermal behavior of the polyurethanes was examined by DSC, using a Perkin-Elmer DSC-7 calibrated with indium. DSC data were obtained from samples of 4-6 mg at heating/cooling rates of 10 °C min-1 under a nitrogen flow. The glass transition temperatures were determined at a heating rate of 20

°C min-1 from rapidly melt-quenched polymer samples. Thermogravimetric analyses (TGAs) were performed under nitrogen atmosphere (flow rate 100 mL min-1) with a Universal V4.3A TA Instrument at a heating rate of 10 °C min-1. The polymerization reactions were performed in absence of humidity under an inert atmosphere. All glassware was heated overnight at 80 °C before use and, after assembly, was further heated under vacuum to eliminate the surface moisture. The diol sugar-based monomers (Scheme 1), 2,3,4-tri-O-benzyl-L-arabinitol (ArBn) and 2,3,4-tri-Omethyl-L-arabinitol (ArMe), were dried under high vacuum for at least 3 days. 1,6-Hexamethylene diisocyanate (HMDI) was stored at 4 °C and 2,2′-dithiodiethanol (DiT) at room temperature, both being handled under an inert atmosphere. Anhydrous tetrahydrofuran (THF) and N,Ndimethylacetamide (DMAc) polymerization solvents were further dried to eliminate residual water. THF was refluxed in the presence of sodium, with benzophenone as indicator, and was freshly distilled prior to use. DMAc was vacuum distilled and stored over molecular sieves in a desiccator for not more than a week before use. The other reactants and reagents for the polymerizations were stored in a desiccator under inert atmosphere until required. Polyurethane PU(DiT-HMDI). In a typical procedure, 2,2′-dithiodiethanol (DiT, 560 µL, 4.15 mmol) was loaded in a round-bottom flask with an inlet of argon/vacuum. The system was treated with three cycles of vacuum-argon before the addition, via cannula, of dried DMAc (2 mL). The mixture was stirred to homogenization, and the diisocyanate (HMDI, 670 µL, 4.15 mmol) was added under an argon atmosphere, followed by the catalyst (dibutyltin dilaurate, one drop). The polymerization solution was stirred for 3 h under an argon atmosphere at room temperature. tert-Butanol (1 mL) was added, the mixture was stirred for 30 min, and the solution was added dropwise into cold diethyl ether (150 mL), where the polymer PU(DiT-HMDI) precipitated. The polyurethane was purified by redissolution in a small volume of chloroform (2 mL) and reprecipitation in diethyl ether. The pure polymer (white solid) was dried under vacuum for 2 days and stored in a desiccator (1.23 g, 92% yield): Mw 29200; Mn 22400; Mw/Mn 1.3. Intrinsic viscosity: 0.13 dL · g-1. IR: ν (cm-1) 3319 (N-H), 2935, 2858 (C-H aliph), 1680 (CdO urethane), 1528 (N-H urethane). 1H NMR (CDCl3, 500 MHz): δ (ppm) 4.95 (bs, 2H, N-H), 4.17 (bs, 4H, SCH2CH2O), 3.02 (bs, 4H, CH2-a), 2.95 (bs, 4H, SCH2CH2O), 1.38 (bs, 4H, CH2-b), 1.23 (bs, 4H, CH2-c). 13C NMR (CDCl3, 125 MHz): δ (ppm) 156.46 (CdO), 62.05 (SCH2CH2O), 40.65 (CH2-a), 37.69 (SCH2CH2O), 29.80 (CH2-b), 26.36 (CH2-c). Anal. Calcd for C12H22N2O4S2: C, 44.70; H, 6.88; N, 8.69; S, 19.89. Found: C, 44.37; H, 6.69; N, 8.72; S, 20.08. Copolyurethane PU[(ArBn80-DiT20)-HMDI]. This copolymer was prepared from 2,3,4-tri-O-benzyl-L-arabinitol (ArBn; 0.211 g, 0.5

Polyurethanes Derived from L-Arabinitol mmol), 2,2′-dithiodiethanol (17 µL, 0.125 mmol), and HMDI (101 µL, 0.625 mmol) in dried-distilled THF (1 mL). The pure polymer (white amorphous solid) was dried under vacuum for 2 days and stored in a desiccator (0.36 g, quantitative yield): [R]D -1.4° (c 1.0, dimethylsulfoxide); Mw 160100; Mn 72300; Mw/Mn 2.2. Intrinsic viscosity: 0.27 dL · g-1. IR: ν (cm-1) 3329 (N-H), 1696 (CdO urethane), 1524 (N-H urethane), 1097 (C-O-CH2Ph), 735, 696 (ar). 1H NMR (CDCl3, 500 MHz): δ (ppm) 7.29 (m, 15H, 3 Ph), 5.22, 5.02 (d and bs, 4H, N-H), 4.71-4.21 (m, 14H, H-1/H-5/3 OCH2Ph/SCH2CH2O), 3.86 (bs, 2H, H-2/H-4), 3.77 (bs, 1H, H-3), 3.11 (bs, 4H, CH2-a), 2.90 (bs, 4H, SCH2CH2O), 1.46 (bs, 4H, CH2-b), 1.30 (bs, 4H, CH2-c). 13C NMR (CDCl3, 125 MHz): δ (ppm) 156.40, 156.29, 156.22 (CdO), 138.35, 138.12, 138.06 (3C, Ph), 128.32, 128.16, 127.94, 127.87, 127.73, 127.64 (15C, Ph), 78.98 (C-3), 77.25, 77.12 (C-2/C-4), 74.65, 73.10, 71.99 (3 OCH2Ph), 63.46, 62.86 (C-1/C-5), 62.38 (SCH2CH2O), 40.88 (CH2a), 37.82 (SCH2CH2O), 29.82 (CH2-b), 26.27 (CH2-c). Anal. Calcd for (C34H42N2O7)78(C12H22N2O4S2)22: C, 65.87; H, 7.13; N, 5.27; S, 2.65. Found: C, 65.82; H, 7.50; N, 5.67; S, 2.74. Copolyurethane PU[(ArBn50-DiT50)-HMDI]. This copolymer was prepared from 2,3,4-tri-O-benzyl-L-arabinitol (ArBn; 0.37 mg, 0.88 mmol), 2,2′-dithiodiethanol (119 µL, 0.88 mmol), and HMDI (283 µL, 1.76 mmol) in dried-distilled DMAc (2 mL). The reaction solvent was, in this case, DMAc, to ensure the complete solubility of the new copolymer synthesized. The pure polymer (white solid) was dried under vacuum for 2 days and stored in a desiccator (0.76 g, 95% yield): [R]D -2.2° (c 1.0, dimethylsulfoxide); Mw 19300; Mn 12900; Mw/Mn 1.5. Intrinsic viscosity: 0.25 dL · g-1. IR: ν (cm-1) 3327 (N-H), 1692 (CdO urethane), 1531 (N-H urethane), 1067 (C-O-CH2Ph), 734, 697 (ar). 1 H NMR (CDCl3, 500 MHz): δ (ppm) 7.26 (m, 15H, 3 Ph), 5.28, 5.08 (2 bs, 4H, N-H), 4.80-4.15 (m, 14H, H-1/H-5/3 OCH2Ph/ SCH2CH2O), 3.82 (bs, 2H, H-2/H-4), 3.74 (bs, 1 H, H-3), 3.09 (bs, 4H, CH2-a), 2.88 (bs, 4H, SCH2CH2O), 1.43 (bs, 4H, CH2-b), 1.27 (bs, 4H, CH2-c). 13C NMR (CDCl3, 125 MHz): δ (ppm) 156.23 (CdO), 138.20, 137.96 (3C, Ph), 128.23, 128.09, 127.85, 127.56 (15C, Ph), 78.89 (C-3), 77.12, 76.98 (C-2/C-4), 74.67, 73.06, 71.91 (3 OCH2Ph), 63.20, 62.66 (C-1/C-5), 62.32 (SCH2CH2O), 40.78 (CH2-a), 37.75 (SCH2CH2O), 29.75 (CH2-b), 26.24 (CH2-c). Anal. Calcd for (C4H9NO)5(C34H42N2O7)48(C12H22N2O4S2)52: C, 59.62; H, 7.35; N, 7.08; S, 6.74. Found: C, 59.63; H, 7.37; N, 7.56; S, 6.80. Copolyurethane PU[(ArMe80-DiT20)-HMDI]. This copolymer was prepared from 2,3,4-tri-O-methyl-L-arabinitol (ArMe, 0.194 g, 1.0 mmol), 2,2′-dithiodiethanol (34 µL, 0.25 mmol), and HMDI (202 µL, 1.25 mmol) in dried-distilled THF (2 mL). The pure polymer (white amorphous solid) was dried under vacuum for 2 days and stored in a desiccator (0.445 g, quantitative yield). [R]D -4.1° (c 1.0, dimethylsulfoxide); Mw 135700; Mn 70600; Mw/Mn 1.9. Intrinsic viscosity: 0.58 dL · g-1. IR (cm-1): 3329 (N-H), 1694 (CdO urethane), 1531 (N-H urethane). 1H NMR (CDCl3, 500 MHz): δ (ppm) 5.21, 5.03 (2 bs, 4H, N-H), 4.56-4.00 (m, 8H, H-1/H-5/SCH2CH2O), 3.63 (bs, 1H, H-2), 3.52 (bs, 1H, H-4), 3.50, 3.47, 3.44 (3 s, 9H, 3 OCH3), 3.35 (bs, 1H, H-3), 3.19 (bs, 4H, CH2-a), 2.95 (t, 4H, J ) 6.5 Hz, SCH2CH2O), 1.53 (bs, 4H, CH2-b), 1.36 (bs, 4H, CH2-c). 13C NMR (CDCl3, 125 MHz): δ (ppm) 156.49, 156.35 (CdO), 79.77 (C-3), 78.33, 77.24 (C2/C-4), 63.10, 62.08 (C-1/C-5), 62.45 (SCH2CH2O), 60.83, 59.08, 57.75 (3 OCH3), 40.91 (CH2-a), 37.86 (SCH2CH2O), 29.85 (CH2-b), 26.28 (CH2-c). Anal. Calcd for (C16H30N2O7)80 (C12H22N2O4S2)20: C, 51.51; H, 8.08; N, 7.90; S, 3.62. Found: C, 51.97; H, 8.26; N, 7.71; S, 3.68. Copolyurethane PU[(ArMe50-DiT50)-HMDI]. This copolymer was prepared from 2,3,4-tri-O-methyl-L-arabinitol (ArMe, 0.194 g, 1.0 mmol), 2,2′-dithiodiethanol (136 µL, 1.0 mmol), and HMDI (324 µL, 2.0 mmol) in dried-distilled DMAc (2 mL). The pure polymer (white solid) was dried under vacuum for 2 days and stored in a desiccator (0.700 g, quantitative yield). [R]D -0.4° (c 1.0, dimethylsulfoxide); Mw 16000; Mn 10800; Mw/Mn 1.5. Intrinsic viscosity: 0.34 dL · g-1. IR (cm-1): 3321 (N-H), 1682 (CdO urethane), 1531 (N-H urethane). 1 H NMR (DMSO-d6, 500 MHz): δ (ppm) 7.14 (bs, 4H, N-H), 4.40-3.80 (m, 8H, H-1/H-5/SCH2CH2O), 3.48 (bs, 1H, H-2), 3.40-3.20


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(m, 11H, H-3, H-4, 3 OCH3), 2.95 (m, 8H, CH2-a, SCH2CH2O), 1.37 (bs, 4H, CH2-b), 1.22 (bs, 4H, CH2-c). 13C NMR (CDCl3, 125 MHz): δ (ppm) 156.63, 156.49, 156.36, 156.06 (CdO), 79.75 (C-3), 79.12 (C-4), 78.80 (C-2) 63.28 (C-1), 62.52 (C-5), 62.04 (SCH2CH2O), 60.31, 58.95, 57.71 (3 OCH3), 40.66 (CH2-a), 37.66 (SCH2CH2O), 29.80 (CH2-b), 26.41 (CH2-c). Anal. Calcd for (C4H9NO)3(C16H30N2O7)51(C12H22N2O4S2)49: C, 49.61; H, 7.86; N, 8.73; S, 8.52. Found: C, 49.53; H, 7.84; N, 9.20; S, 8.90. Copolyurethane PU[(ArOH80-DiT20)-HMDI]. This copolymer was prepared from commercial L-arabinitol (ArOH, 0.456 g, 3.0 mmol), 2,2′-dithiodiethanol (102 µL, 0.75 mmol), and HMDI (606 µL, 3.75 mmol) in dried-distilled DMAc (2 mL) at -17 °C. The reaction mixture was stirred for 1 h, allowed to warm up to 5 °C, and then stirred for a further 30 min. The reaction was worked up as usual. The pure polymer (white solid) was dried under vacuum for 2 days and stored in a desiccator (0.976 g, 81% yield). Mw 10700; Mn 5600; Mw/Mn 1.9. Anal. Calcd for (C13H24N2O7)80(C12H22N2O4S2)20: C, 47.93; H, 7.42; N, 8.73; S, 4.00. Found: C, 54.32; H, 8.97; N, 10.11; S, 0.67. Copolyurethane PU[(ArOH50-DiT50)-HMDI]. This copolymer was prepared from commercial L-arabinitol (ArOH, 0.456 g, 3.0 mmol), 2,2′-dithiodiethanol (408 µL, 3.0 mmol), and HMDI (969 µL, 6.0 mmol) in dried-distilled DMAc (2 mL) at -17 °C. The reaction mixture was stirred for 1 h, allowed to warm up to 5 °C, and then stirred for a further 30 min. The reaction was worked up as usual. The pure polymer (white solid) was dried under vacuum for 2 days and stored in a desiccator (0.843 g, 44% yield). Mw 8000; Mn 4200; Mw/Mn 1.9. Anal. Calcd for (C13H24N2O7)50(C12H22N2O4S2)50: C, 46.71; H, 7.21; N, 8.72; S, 9.98. Found: C, 54.10; H, 8.54; N, 10.83; S, 4.32. Chemical Degradation of Homo- and Copolymers with Glutathione. Preparation of Polymer Disks. Five polymer disks (140 ( 10 µm of thickness) were made from each polymer studied. The disks were prepared by the application of 10 ton cm-2 pressure on powdered polymer (20 ( 5 mg) for 5 min, at 25 °C. The disks were then dried at 45 °C for 3 days. Degradation Conditions. Each polymer disk was submerged in a reduced glutathione solution (10 mL, 0.1 M, 1 mmol), at pH 7.02. An argon flow was passed through the solution for 5 min, and the vial was then sealed and heated at 37 °C for an exact period of time. The degradation experiments were quenched by methylation of the free thiol groups generated to stabilize the degraded polymer fragments. Quenched Degradation Experiments. The vial content was poured into a flask provided with a stirring bar. THF (10 mL), sodium hydrogen carbonate (252 mg, 3 mmol), and methyl iodide (0.44 mL, 7 mmol) were added sequentially. The mixture was stirred at 25 °C for 5 h. The liquid phase was eliminated, and the disks were washed with distilled water (3 × 10 mL) and then dried. The molecular weights (Mn and Mw) and polydispersity indexes (Mw/Mn) of the degraded polymers were studied by gel permeation chromatography.

Results and Discussion The synthesis of the novel linear copolyurethanes was carried out by reaction of 1,6-hexamethylene diisocyanate (HMDI) and mixtures of 2,2′-dithiodiethanol (DiT) and 2,3,4-tri-O-benzylL-arabinitol (ArBn) or 2,3,4-tri-O-methyl-L-arabinitol (ArMe), with higher or equal proportion of the sugar-based monomer related to DiT (Scheme 1). The novel homopolyurethane PU(DiT-HMDI) was also prepared as a model compound (Scheme 2). The homopolymers PU(ArBn-HMDI) and PU(ArMeHMDI) were prepared from ArBn and ArMe, respectively, as described in earlier papers of our group.22,23 The yields and physical properties of the homo- and copolymers are shown in Table 1. The polymerization solvent was THF when the L-arabinitol-based monomer was the main diol component in the polyurethanes. The use of THF may lead to slightly soluble semicrystalline polyurethanes when the DiT ratio increases. Hence, the precipitation of the copolymers in

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Table 1. Yields and Some Physical Properties of the Homo- and Copolyurethanes polymer

yield (%)

[R]Da

PU(DiT-HMDI) PU(ArBn-HMDI)d PU[(ArBn80-DiT20)-HMDI] PU[(ArBn50-DiT50)-HMDI] PU(ArMe-HMDI)e PU[(ArMe80-DiT20)-HMDI] PU[(ArMe50-DiT50)-HMDI] PU[(ArOH80-DiT20)-HMDI] PU[(ArOH50-DiT50)-HMDI]

92 95 quant 95 95 quant quant 81 44

0 -0.3° -1.4° -2.2° -4.2° -4.1° -0.4

[η]

b

Mnc

Mwc

22400 29200 72300 12900 22000 70600 10800 5600 4200

29200 52100 160100 19300 56800 135700 16000 10700 8000

(dL/g)

0.13 0.39 0.27 0.25 0.67 0.58 0.34

Mw/Mn

c

1.3 1.8 2.2 1.5 2.5 1.9 1.5 1.9 1.9

a Specific rotation in degrees, c 1, in dimethylsulfoxide b Intrinsic viscosity in dL/g determined in dichloroacetic acid at 25 °C. c Determined by GPC analysis against polystyrene standards using NMP as mobile phase. d Data published in ref 22. e Data published in ref 23.

Table 2. Copolymer Composition Calculated by Elemental Analysis and 1H NMR Spectroscopy diol molar ratio Ar/DiT

elemental analysis c

calculated polymer

feed

PU(DiT-HMDI) PU(ArBn-HMDI) d PU[(ArBn80-DiT20)-HMDI] PU[(ArBn50-DiT50)-HMDI] PU(ArMe-HMDI) e PU[(ArMe80-DiT20)-HMDI] PU[(ArMe50-DiT50)-HMDI] PU[(ArOH80-DiT20)-HMDI] PU[(ArOH50-DiT50)-HMDI]

0:100 100:0 80:20 50:50 100:0 80:20 50:50 80:20 50:50

theoretical

experimental

1

elemental analysisa

H NMR analysisb

0:100 100:0 78:22 48:52 100:0 80:20 51:49

0:100 100:0 73:27 45:55 100:0 76:24 57:43

C (%)

H (%)

N (%)

S (%)

C (%)

H (%)

N (%)

S (%)

44.70 68.09 66.20 59.55 52.99 51.51 49.53 47.93 46.71

6.88 7.23 7.13 7.45 8.37 8.08 7.85 7.42 7.21

8.69 4.67 5.22 7.09 7.95 7.90 8.74 8.73 8.72

19.89 0.00 2.39 6.49 0.00 3.62 8.70 4.00 9.98

44.37 68.05 65.82 59.63 52.79 51.97 49.53 54.32 54.10

6.69 7.34 7.50 7.37 7.91 8.26 7.84 8.97 8.54

8.72 4.95 5.67 7.56 8.39 7.71 9.20 10.11 10.83

20.08 0.00 2.74 6.80 0.00 3.68 8.90 0.67 4.32

a Molar composition of diol monomers in the polymers determined from elemental microanalysis. b Molar composition of diol monomers determined from 1H NMR spectra. c According to the molar ratio of diol monomers in the feed. d Data published in ref 22. e Data published in ref 23.

the reaction media would stop the polymerization process as the DiT monomer reduces the solubility and increases the crystallinity of the new macromolecules. Dry DMAc was used in such situations. The recovered polymers were isolated with excellent yields (over 90% in most cases). The highest Mw values were found for the copolymers with the highest percentage of the sugar diol residue in their backbones, with enhanced solubilities in the media. Several attempts were made to synthesize copolyurethanes using fully deprotected L-arabinitol as one of the comonomers. The reactions proceeded at low temperature (from -17 to 5 °C) for 1.5 h. The new materials presented low Mw (Table 1), and their 1H NMR and elemental microanalyses suggested that cross-linking reactions took place. The signals of 1H and 13 C NMR from unreacted DiT monomer were found in the NMR spectra, and the incorporation into the polymer backbone was scant. From these data it was concluded that the DiT monomer is less reactive than L-arabinitol. Therefore, the deprotected L-arabinitol-based copolymers were not further investigated. The copolymer compositions were studied by elemental microanalyses and 1H NMR. These data (Table 2) reveal that the copolymer content in alditols and 2,2′-dithiodiethanol units is in all cases very similar to that of their corresponding feed. The calculated monomer ratio data are those values that lead to the best agreement with the experimental values from 1H NMR and elemental microanalyses. From the percentage of the different elements (by elemental microanalyses), together to the sulfur content (Table 2, it is possible to know the ratio of the DiT monomer incorporated in the polymer backbone. The ratio was established between the two possible repeating units in the copolymer, DiT-HMDI and sugar-HMDI (ArMe-HMDI or ArBn-HMDI). The calculated monomer ratio fluctuated within (2% that of the feed, underlining the excellent control over the final copolymer composition. The theoretical elemental microanalysis data displayed were calculated according to the

feed composition in diol monomers. The copolymer composition was also calculated by 1H NMR spectroscopy. The monomer ratio obtained by 1H NMR was the result of comparing the integrals of the peaks assigned to H-3 from the L-arabinitol residue in the polymer (∼3.77 ppm) with those of the protons -CH2S- from the DiT monomer (∼2.90 ppm). Scrutiny of these results also highlights that the actual copolymer composition was quite similar to theoretical ones (see Table 2). The thermal properties of the polyurethanes are displayed in Table 3. The PU-(DiT-HMDI) homopolymer exhibits a highly crystalline structure, with Tm ) 145 °C and a melting enthalpy of 62 J/g. In accordance with this, the copolymers with high proportion of DiT monomer in their composition are all semicrystalline (sugar-DiT ratio: 50:50), although the ∆Hm decreases substantially compared with the homopolymer. A reduction in DiT monomer to values close to 20% (mol ratio) leads to the loss of crystallinity. The presence of the sugar units (ArBn and ArMe) is responsible for the amorphous nature of the 80:20 copolymers. As expected, for the semicrystalline copolymers, the smaller size of the methyl group compared with the benzyl ones leads to a higher crystalline fraction in the polymeric structure, as confirmed by the higher ∆Hm values. The Tg values of both series of copolymers are consistent with the Tgs of their related homopolymers. These values suggest that the ArBn-based copolymers (Tgs ∼ 18 °C) are stiffer than the ArMe-based ones (Tgs ) 11 °C). To understand the thermal decomposition of the copolymers prepared, it is necessary to know the general behavior of polymers having disulfide bonds in their general structure. Proteins are the main natural polymers with disulfide bonds between two cysteine units. Thus, because of the importance of proteins, there is deep knowledge on the thermal decomposition of their disulfide bonds.32-34 This type of degradation is one of the reasons for the irreversible loss of enzymatic activity at high temperature. It is well-known that thermal cleavage of

Polyurethanes Derived from L-Arabinitol


273

Table 3. Thermal Properties of the Homo- and Copolyurethanes DSC a

Tm (°C)

polymer

∆H (J/g)

b

Tc ∆H (°C) (J/g)b

PU(DiT-HMDI) 147 63 105 -44 PU(ArBn-HMDI) PU[(ArBn80-DiT20)HMDI] 3/7 PU[(ArBn50-DiT50)HMDI] 50/90 PU(ArMe-HMDI) PU[(ArMe80-DiT20)HMDI] PU[(ArMe50-DiT50)HMDI] 50/81/105 4/9/14

c

Tm (°C) 145

TGA ∆H (J/g)c 62

88/104 3/1

d

Tm (°C)

∆H Tg Td0 (J/g)d (°C) Tg/Tme (°C)f

145 69

65 12

83/99

8/3

80/103 19/1

1 36 17 19 30 11 11

1.5 1.1 1.2 1.2

266 319 280 280 278 253 261

Td (°C)g

∆W (%)h

268/359/450 360/456 280/351/447 288/351/457 297/366/469 254/312/360/459 224/275/357/463

67/18/11 96/2 17/79/3 33/62/4 64/12/24 20/46/23/11 5/52/30/8

First heating (10 °C/min). Cooling trace after melting (10 °C/min). Second heating (20 °C/min), after quenching from melting to -40 °C. d After annealing at 65 °C, for 48 h. e K/K. f Onset of decomposition. g Decomposition temperatures measured at the peaks of the derivative curves; major peaks in bold; decomposition of disulfide bonds underlined. h Loss weight at the end of the decomposition step. a

b

Figure 1. Comparative curves of thermal degradation under inert atmosphere of the model PU(ArBn-HMDI) and PU(DiT-HMDI) homopolymers with PU[(ArBn-DiT)-HMDI] copolymers: weight loss (%) vs temperature.

a disulfide bond in proteins is the result of β-elimination from the cystine residue and could lead to thiocysteine residues (and a possible loss of elemental sulfur), cysteine residues, and dehydroalanine.35 Volkin and Klibanov32 studied more than a dozen proteins at 100 °C, and found that they all underwent β-elimination of disulfides, at similar rates. The process turned out to be relatively independent of both the primary structure and the elements of higher structures remaining in the proteins at 100 °C. The products derived from the thermal cleavage of cystine were confirmed by mass spectrometry.33 The thermal degradation of poly(methyl methacrylate)poly(styrene disulfide) blends was investigated by Ganesh et al.36 using thermogravimetry and direct pyrolysis-mass spectrometric (DP-MS) analysis. Some of the products produced in the DP-MS analysis of the blends were S2, styrene, and styrene sulfide, among others. This trend backs up the notion that β-elimination is a general mechanism of disulfide compounds, as observed in the case of cystine, with possible elimination of elemental sulfur. Thus, it is possible to infer that the new synthesized copolyurethanes undergo a similar thermal degradation pattern. Figures 1 and 2 represent the TGA curves of PU(ArBnHMDI) and PU(DiT-HMDI) homopolymers and their copolymers at different feed ratios: PU[(ArBn80-DiT20)-HMDI] and PU[(ArBn50-DiT50)-HMDI]. The TG curve of PU(DiT-HMDI) shows a three-step degradation process. The first and main step occurred at 268 °C, with an associated weight loss of over 65%. The degradation may involve cleavage of the disulfide bond by β-elimination and the formation of elemental sulfur and terminal alkenes. In contrast, the PU(ArBn-HMDI) homopolymer shows

c

Figure 2. Comparative curves of thermal degradation under inert atmosphere of the model PU(ArMe-HMDI) and PU(DiT-HMDI) homopolymers with PU[(ArMe-DiT)-HMDI] copolymers: weight loss (%) vs temperature.

Figure 3. Chemical degradation of the model PU(DiT-HMDI) homopolymer and ArBn-based copolyurethanes.

an almost one-step degradation at higher temperature (360 °C), with a weight loss of above 95%. The TG curves of both the ArBn- and ArMe-based copolymers exhibited a mixed trend with several degradation steps between those of the related homopolymers. Evidently, each copolymer possessed a degradation step (shown underlined in Table 3) that can be assigned to the cleavage of the disulfide bond, as in the case of the homopolymer PU(DiT-HMDI). As expected, the weight loss associated with the degradation of disulfide bond increased with the DiT ratio (from 20 to 50%) in the polymer; it increased from 17 to 33% for the ArBn-based copolymer and from 20 to 52% for the ArMe-based one. The decomposition of disulfide bond occurred at different temperature for different polymer. Generally speaking, the copolymers exhibited an increase of the decomposition temperature, when compared to the homopolymer PU(DiT-HMDI). This is not surprising since the

274


de Paz et al.

Figure 4. Chemical degradation of the model PU(DiT-HMDI) homopolymer and ArMe-based copolyurethanes.

sugar-based homopolymers were more thermoresistant than the DiT-based one. However, the pyrolysis of the disulfide bond for PU[(ArMe80-DiT20)-HMDI] occurred at lower temperature (254 °C), which can be mainly assigned to its amorphous structure. The degradation behavior of the new copolymers under physiological conditions (at 37 °C and pH 7.02) was studied and their trends were compared with that of the model homopolymer PU-(DiT-HMDI). No significant losses in the weight of the samples were observed in these experiments, which can be explained by the nonsolubility of the degradation products in the incubation media. Degradation of these polyurethanes was monitored by GPC to determine the changes in molecular weight that the samples undergo during the incubation period. Figures 3 and 4 and Table 4 show the evolution of the weight-average molecular weights (Mw) during the hydrolysis of these polymers. Degradation of the model polyurethane [PU(DiT-HMDI)] is characterized by a rapid decrease in molecular weight during the first period of incubation, followed by a second period in which degradation proceeds at a lower rate. However, the degradation rates of the copolymers keep almost constant all along the incubation period. The SEM analysis of samples before and after degradation provided valuable information about the degradation mechanism. Micrographs of the original PU[(ArMe80-DiT20)-HMDI] disk and the disk incubated for 5 days at pH 7.0 are shown in Figure 5. The initial disk thickness of about 140 µm reduced to less than half upon degradation, and numerous holes are observable on the disk, indicating that the material underwent attack in the bulk. We can assume that the small size of the tripeptide glutathione (the most abundant low-molecular-weight biological thiol) facilitates its dispersion into the polymer disk all along the trials with degradation effects not only over the surface of the polymer disks but also in their inner part. A large amount of material appears attached on the surface of the disk in the form of granules; this is thought to comprise the nonsoluble fragments that are generated upon hydrolysis. It seems, therefore, that no weight loss is observed because the degraded material is not released into the incubation medium. The glutathione-mediated degradation pathway of 2,2′dithiodiethanol-based polyurethanes is shown in Scheme 3. As

Figure 5. SEM micrographs of the PU[(ArMe80-DiT20)-HMDI] disk before (a) and after (b) incubation at pH 7.0 for 5 days.

mentioned before, glutathione exists in reduced (GSH) and oxidized (glutathione disulfide, GSSG) states, where the GSH/ GSSG system is the major redox couple in animal cells.25 Disulfide bonds are prone to rapid cleavage under a reductive environment through the fast and readily reversible thioldisulfide exchange reactions.26,27 In the present case, it is assumed that the lyses of the polymeric disulfide bonds followed the same mechanism as mentioned above (Scheme 3). The disulfide groups from the polymer were reduced by GSH leading to the degradation of the polymer backbones and the simultaneous formation of glutathione disulfide (GSSG). The degraded polymeric fragments were later stabilized by entrapping the newly generated free thiol groups as methyl thioether by

Table 4. Reductive Degradation Rates and Related Parameters remaining molecular weight (%)a polymer

Tm (°C)

∆H (J/g)

meq SS/g

2 days

4 days

10 days

20 days

35 days

PU(DiT-HMDI) PU[(ArMe50-DiT50)HMDI] PU[(ArBn50-DiT50)HMDI] PU[(ArMe80-DiT20)HMDI] PU[(ArBn80-DiT20)HMDI]

145 80/103 83/99

65 19/1 8/3

3.10 1.43 1.15 0.56 0.41

30.03 97.02 94.23 83.96 90.08

4.92 94.96 90.25 74.21 77.99

89.24 78.28 49.40 47.43

81.21 58.35 22.87 15.51

72.32 28.45 17.86 8.12

a

Based on Mw.

Polyurethanes Derived from L-Arabinitol


275

Scheme 3. Degradation of 2,2′-Dithiodiethanol-Based Polyurethanes Mediated by Reduced Glutathione under Physiological Conditions

treatment of the degraded disks with MeI in aqueous THF media, before being studied by GPC and SEM. The value of milliequivalents of disulfide groups per gram of polymer (from 3.10 to 0.41, Table 4) was assumed to be a key parameter in their kinetics. As expected, the PU(DiT-HMDI) homopolymer presented a very high degradation rate (a reduction in its Mw of over 95% in four days). However, in the case of the copolymers, the crystallinity also plays an important role in the degradation rate. So, the copolymers with ∼50% DiT molar ratio presented longer decomposition times than the copolymers with lower percentages of DiT diol monomer (Figures 3 and 4). The general trend was that the higher the fusion enthalpy, the lower the degradation rate. Copolymers PU[(ArBn50-DiT50)-HMDI] and PU[(ArMe50-DiT50)-HMDI] have well-packed semicrystalline regions (with the DiT-HMDI repeating unit involved) that are of difficult access to the reducing agent (the hydrophilic reduced glutathione). In contrast, the amorphous copolymers with low DiT-HMDI repeating unit contents are more easily cleaved, despite the lower disulfide ratio. Their DiT-HMDI repeating units do not pack into semicrystalline segments and are, thus, more accessible to the reducing agent. Therefore, the amorphous copolymers PU[(ArBn80DiT20)-HMDI] and PU[(ArMe80-DiT20)-HMDI] are those with enhanced degradation trends.

Conclusions A batch of novel linear copolyurethanes have successfully been synthesized by reaction of 1,6-hexamethylene diisocyanate (HMDI) and mixtures of 2,2′-dithiodiethanol (DiT) and the sugar-based diols 2,3,4-tri-O-benzyl-L-arabinitol (ArBn) or 2,3,4tri-O-methyl-L-arabinitol (ArMe). The DiT diol acts as a carrier of the reactive segments for future degradations. The excellent agreement between the experimental copolymer compositions and the composition of the monomers in their corresponding

feed was established by elemental analyses and 1H NMR data. The enriched DiT copolymers were semicrystalline, with the highest degree of crystallinity displayed by the PU (DiT-HMDI) homopolymer. The homo- and copolymers were thermally stable up to 220 °C in all cases. The novel homo- and copolymers were degraded at pH 7.02 and 37 °C by the action of the tripeptide glutathione. No weight loss was detected upon degradation due to the nonsolubility of the degraded products in the aqueous incubation medium but the polymer disks were significantly distorted in accordance with the important reductions in Mw recorded by GPC. The degradation pattern of the copolymers depended not only on the dithiodiethanol (DiT) reactive units ratio in the polymer backbone but also on the crystallinity of the final macromolecule. Thus, the highest degradation rates were achieved for the model homopolymer (maximum ratio of DiT units) and for the amorphous copolymers {PU[(ArBn80-DiT20)-HMDI] and PU[(ArMe80-DiT20)HMDI]}. Acknowledgment. We thank the CICYT (Comisioń Interministerial de Ciencia y Tecnologıá) of Spain and the Junta de Andalucıá for financial support (Grants MAT2006-13209-C02 and FQM-02648).

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Glutathione-Mediated Biodegradable Polyurethanes Derived from l

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