Methylated Mono- and Di(ethylene glycol)-Functionalized β-Sheet

We have synthesized methylated mono- and di(ethylene glycol)-functionalized polymers of l-serine and l-cysteine that adopt β-sheet conformations in t...
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Biomacromolecules 2001, 2, 17-21

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Communications Methylated Mono- and Di(ethylene glycol)-Functionalized β-Sheet Forming Polypeptides Jungyeon Hwang and Timothy J. Deming* Departments of Materials and Chemistry, University of California, Santa Barbara, Santa Barbara, California 93106 Received August 10, 2000

We have synthesized methylated mono- and di(ethylene glycol)-functionalized polymers of L-serine and L-cysteine that adopt β-sheet conformations in the solid state: poly(O-(2-(2-methoxyethoxy)ethyl)-L-serine), poly(1); poly(O-(2-(methoxy)ethyl)-L-serine), poly(2); and poly(S-(2-(2-methoxyethoxy)ethoxy)carbonylL-cysteine), poly(3). Of these three polymers, only poly(1) was found to be highly soluble in water independent of pH. Circular dichroism analysis of poly(1) in water or trifluoroethanol at 25 °C revealed that it is in a random conformation, which was unperturbed by changes in pH, buffer, or temperature. However, addition of methanol or acetonitrile to aqueous solutions of poly(1) resulted in a transition to the β-sheet conformation, as found in the solid state. The polymers were synthesized by transition metal catalyzed polymerization of amino acid-N-carboxyanhydrides, prepared from the functionalized amino acids and represent a new class of readily processable β-sheet forming polypeptides. Introduction. Many homopolypeptides adopt stable β-sheet conformations in the solid state.1 As a result of intermolecular H-bonding, these polymers are universally insoluble in water unless their degrees of polymerization are very low (20 residues or less)2 or their side-chain functional groups are ionized by adjustment of pH.3 Both of these exceptions result in solutions in which the polymers adopt predominantly random conformations.2,3 Successful processing of β-sheet forming polypeptides thus requires either the use of harsh denaturing solvents (e.g., trifluoroacetic acid (TFA)) to cast films or pH adjustment to precipitate the polymers in the β-form.3 We have been interested in the self-assembly of block copolypeptides containing distinct secondary structural elements (e.g., R-helices and β-sheets) for potential biomedical applications. To anneal such polymers into regular assemblies, it is desirable to have β-sheet domains that can be gradually assembled in aqueous solution. To this end, we have prepared methylated mono- and di(ethylene glycol)functionalized polymers of L-serine and L-cysteine (see Scheme 1) that adopt β-sheet conformations in the solid state. One of these, poly(O-(2-(2-methoxyethoxy)ethyl)-L-serine), poly(1), was found to dissolve in water as a random coil that could be induced to adopt the β-sheet structure independent of solution pH. Polypeptides composed primarily of β-sheets are notoriously difficult to process. For example, mechanical spinning of silk proteins4 or silklike polypeptides,5 which contain substantial β-sheet content, usually requires the use of strongly denaturing agents to disrupt the interchain associa-

Scheme 1. Synthesis of EG-Modified Amino Acids 1-3

tions in these materials so that the polymer solutions can flow. These solutions, containing polymers in random conformations, are then subjected to extensional shear in addition to a coagulant or diluant that ideally drives the polymer into an ordered β-sheet structure.6 Typically, the rapid association of the chains during coagulation prevents a high degree of ordering and there is a considerable amount of random aggregation in these materials.6 Likewise, homopolymers of amino acids that adopt β-sheet structures (e.g., poly L-valine and poly L-serine) are generally insoluble in all but the most strongly denaturing solvents (e.g., TFA or 8 M LiBr in H2O) and do not lend themselves to straightforward manipulation.7 β-sheet forming polypeptides with ionizable side chains (e.g., poly L-cysteine and poly carboxyalkyl-L-cysteines)3 are soluble in alkaline water, but are

10.1021/bm005597p CCC: $20.00 © 2001 American Chemical Society Published on Web 11/23/2000

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unstructured in solution and must be acidified to adopt the β-conformation, whereupon they precipitate. Polymer processing methods that employ strongly denaturing solvents or pH adjustments are undesirable for formation of ordered self-assemblies from block copolypeptides containing multiple secondary structure domains. For example, nonselective disruption of all secondary structures with a strong denaturant hinders the ability to separately assemble individual domains. Adjustment of pH is also problematic in that other polypeptide domains may also be pH sensitive (e.g., poly(L-glutamic acid)), and thus selectivity in assembly is compromised. We had recently reported that modification of the side chains of L-lysine with short repeats of ethylene glycol (EG) converted polymers of this amino acid from pH sensitive polyelectrolytes into pH insensitive, water-soluble, R-helical polymers.8 It was thought that addition of similar EG modifications to the β-sheet preferring amino acids L-serine and L-cysteine might similarly allow facile aqueous processing of their corresponding β-forming polymers. The EG side chains should provide good water solubility to the polymers, which could then form β-sheet structures upon solvent evaporation or by controlled addition of a solvent that stabilizes the β-conformation. The EG segments could also impart biocompatability to the polymers through formation of an EG coating around the backbone, which would form a “steric barrier” to biological recognition, similar to poly(ethylene glycol) (PEG).9 Experimental Section. General Data. Tetrahydrofuran, hexane, and diethyl ether were dried by passage through a column packed with alumina under nitrogen prior to use.10 Dimethylformamide was dried by passage through a column packed with molecular sieves (4A) under nitrogen prior to use. Chemicals were purchased from commercial suppliers and used without purification. (PMe3)4Co was prepared according to the literature procedure.11 Infrared spectra were recorded on a Perkin-Elmer RX1 FTIR spectrophotometer calibrated using polystyrene film. 1H NMR spectra were recorded on a Bruker AVANCE 200 MHz spectrometer. Molecular weights were obtained by analysis of 3 M LiBr aqueous polymer solutions using a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP (for poly(1): dn/ dc ) 0.118 mL/g). Circular dichroism measurements were carried out on a Olis rapid scanning monochromator at room temperature. The path length of the quartz cell was 1.0 mm, and the concentration of peptide was 0.5 mg/mL. Dynamic light scattering measurements were performed on a Brookhaven Instruments DLS apparatus using a He-Ne 30 mW laser and by measuring the scattered intensity at an angle of 90°. MALDITOF mass spectra were collected using a Thermo BioAnalysis DYNAMO mass spectrometer running in positive ion mode with samples prepared by mixing solutions of analyte in THF with solutions of 2,5-dihydroxybenzoic acid in THF and allowing the mixture to air-dry. Nr-tert-Butyloxycarbonyl-O-(2-(2-methoxyethoxy)ethyl)L-serine. NR-tert-butyloxy-carbonyl-L-serine (4.59 g, 22.3 mmol) was dissolved in N,N-dimethylformamide (100 mL); the solution was then cooled to 0 °C and treated with sodium hydride (1.97 g, 49.2 mmol). 1-Bromo-2-(2-methoxyethoxy)ethane (10.0 g, 49.2 mmol) was added to the solution, and

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the reaction mixture was stirred at ambient temperature for 3 h. The solvent was then removed under reduced pressure at 40 °C bath temperature. The residue was dissolved in water (75 mL) and washed twice with diethyl ether (30 mL each time). The aqueous layer was then acidified to pH 3 with 1 N HCl and then extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO4. and the solvent was removed in vacuo to yield a yellow oil (4.1 g, 63%). FTIR (CHCl3): 1735 (νCO, s), 1712 (νCO, s). 1H NMR (CDCl3): δ 5.63 (d, (CH3)3COC(O)NHCH(CH2OCH2CH2O-CH2CH2OCH3)C(O)OH, 1H), 4.38 (s, (CH3)3COC(O)NHCH(CH2OCH2CH2O-CH2CH2OCH3)C(O)OH, 1H), 4.14-3.53 (m, (CH3)3COC(O)NHCH(CH2OCH2CH2O-CH2CH2OCH3)C(O)OH, 10H), 3.47 (s, (CH3)3COC(O)NHCH(CH2OCH2CH2OCH2-CH2OCH3)C(O)OH, 3H), 1.52 (s, (CH3)3COC(O)NHCH(CH2O-CH2CH2OCH2CH2-OCH3)C(O)OH, 9H). MALDITOF-MS, MH+: 307.34, calcd; 309.19, found. O-(2-(2-Methoxyethoxy)ethyl)-L-serine. NR-tert-Butyloxycarbonyl-O-(2-(2-methoxy-ethoxy)ethyl)-L-serine was used without further purification. This serine derivative (4.13 g, 16.9 mmol) was dissolved in concentrated acetic acid (50 mL). After placing the solution in an ice bath, 1 N HCl (34 mL) was then added and the mixture stirred for 1/2 h. The stirring was continued at ambient temperature for 2 h, and the solution was then concentrated under reduced pressure to yield a yellow oil. The oil was then neutralized with Et3N, and the amine salt was removed by extraction with CH3CN. The insoluble product was collected as white solid (2.4 g, 58%). 1H NMR (D2O): δ 3.87 (m, NH2CH(CH2OCH2CH2OCH2CH2OCH3)C(O)OH, 3H), 3.67-3.60 (m, NH2CH(CH2O-CH2CH2OCH2CH2OCH3)C(O)OH, 8H), 3.34 (s, NH2CH(CH2OCH2CH2OCH2CH2O-CH3)C(O)OH, 3H). 13C {1H} NMR (D2O): δ 173.05 (NH2CH(CH2OCH2CH2OCH2CH2OCH3)C(O)OH), 71.97, 70.87, 70.64, 70.48, 69.80 (NH2CH(CH2OCH2CH2OCH2-CH2OCH3)C(O)OH), 59.10 (NH2CH(CH2OCH2CH2O-CH2CH2OCH3)C(O)OH), 55.67 (NH2CH(CH2OCH2CH2OCH2CH2OCH3)C(O)OH). MALDITOFMS, MH+: 207.22, calcd; 208.48, found. [R]D23 ) -11.2 (c ) 0.05, H2O). O-(2-(2-Methoxyethoxy)ethyl)-L-serine NCA. To O-(2(2-methoxyethoxy)ethyl)-L-serine (0.64 g, 2.6 mmol) was added THF (100 mL) and COCl2 (1.63 mL of a 1.93 M toluene solution), and the mixture was stirred for 5 h at room temperature. The resulting solution was concentrated to give a yellow oil as the crude product (0.49 g, 80%). The oil was crystallized from a tetrahydrofuran, toluene, and hexane mixture (1:4:4) at -30 °C to give the product as a white solid (0.27 g, 45%). FTIR (THF): 1858 cm-1 (νCO, s), 1792 cm-1 (νCO, s). 1H NMR (CDCl3): δ 7.53 (s, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH2CH2OCH3, 1H), 4.40 (t, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH2CH2OCH3, 1H), 3.90 (d, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH2CH2OCH3, 2H), 3.70-3.55 (m, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH2CH2OCH3, 8H), 3.41 (s, RC(H)C(O)OC(O)NH, R ) -CH2OCH2-CH2OCH2CH2OCH3, 3H). 13C {1H} NMR (CDCl3): δ 169.82 (RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH2CH2OCH3), 153.63 (RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2-OCH2CH2OCH3), 72.75, 72.26, 71.76, 71.32, 71.07 (RC(H)C(O)-

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OC(O)NH, R ) -CH2O-CH2CH2OCH2CH2OCH3), 59.95 (RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2O-CH2CH2OCH3), 59.78 (RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH2CH2OCH3) [R]D23 ) -37.6 (c ) 0.017, THF). Poly(O-(2-(2-methoxyethoxy)ethyl)-L-serine), Poly(1). O-(2-(2-methoxyethoxy)ethyl)-L-serine NCA (120 mg, 0.52 mmol) in DMF (2 mL) was mixed with (PMe3)4Co (3.8 mg, 0.010 mmol) in THF (0.5 mL) and stirred for 18 h. The polymer was precipitated from this solution by addition to hexane (20 mL). The polymer was dissolved in H2O (5 mL) and dialyzed to remove impurities and then freeze-dried to give the product as a white solid (70 mg, 71%). FTIR (KBr): 1631 cm-1 (amide I, s br), 1523 cm-1 (amide II, s br). 1H NMR (D2O): δ 8.45 (d, -(NHCH(CH2OCH2CH2OCH2CH2OCH3)C(O))n-, 1H), 4.61 (m, -(NHCH(CH2OCH2CH2OCH2CH2OCH3)C(O))n-, 1H), 3.80 (br s, -(NHCH(CH2O-CH2CH2OCH2CH2OCH3)C(O))n-, 2H), 3.673.61 (br m, -(NHCH(CH2OCH2CH2O-CH2CH2OCH3)C(O))n-, 8H), 3.36 (s, -(NHCH(CH2OCH2CH2OCH2CH2OCH3)C(O))n-, 3H). 13C {1H} NMR (D2O): δ 174.23 (-(NHCH(CH2OCH2CH2OCH2CH2OCH3)C(O))n-), 72.06, 71.15, 70.75, 70.59 (-(NHCH(CH2OCH2CH2OCH2CH2OCH3)C(O))n-), 59.15 (-(NHCH(CH2OCH2CH2OCH2CH2OCH3)C(O))n-), 54.52 (-(NHCH(CH2OCH2CH2O-CH2CH2OCH3)C(O))n-). [R]D23 ) -28.3 (c ) 0.012, H2O). GPC: Mn ) 17 000, Mw/Mn ) 1.22). N r -tert-Butyloxycarbonyl-O-(2-(methoxy)ethyl)- L serine. NR-tert-Butyloxycarbonyl-L-serine (4.59 g, 22.3 mmol) was dissolved in DMF (100 mL), and the resulting solution was then cooled to 0 °C and treated with sodium hydride (1.97 g, 49.2 mmol). 2-Bromoethyl methyl ether (6.84 g, 49.2 mmol) was then added to the solution, and the reaction mixture was stirred at ambient temperature for 3 h. The solvent was then removed under reduced pressure at 40 °C bath temperature. The residue was dissolved in water (75 mL) and washed twice with diethyl ether (30 mL each time). The aqueous layer was then acidified to pH 3 with 1 N HCl and then extracted with ethyl acetate. The organic layer was separated and dried over anhydrous MgSO4, and the solvent was removed in vacuo to yield the product as a yellow oil (2.57 g, 43.8%). This compound was used for subsequent work without further purification FTIR (CH2Cl2): 1714 cm-1 (νCO, s). 1H NMR (CDCl3): δ 5.83 (d, (CH3)3COC(O)NHCH(CH2O-CH2CH2OCH3)C(O)OH, 1H), 4.54 (d, (CH3)3COC(O)NHCH(CH2OCH2CH2OCH3)-C(O)OH, 1H), 4.063.85 (m, (CH3)3COC(O)NHCH(CH2OCH2CH2OCH3)C(O)OH, 2H), 3.66-3.76 (m, (CH3)3COC(O)NHCH(CH2OCH2CH2OCH3)C(O)OH, 4H), 3.48 (s, (CH3)3COC(O)NHCH(CH2OCH2CH2OCH3)C(O)OH, 3H), 1.55 (s, (CH3)3COC(O)NHCH(CH2OCH2CH2OCH3)C(O)OH, 9H). MALDITOF-MS, MH+: 263.29, calcd; 263.03, found. O-(2-(Methoxy)ethyl)-L-serine. NR-tert-butyloxycarbonyl-O-(2-(methoxy)-ethyl)-L-serine (2.57 g, 9.76 mmol) was dissolved in glacial acetic acid (50 mL). 1 N HCl (34 mL) was added to this solution which was cooled in an ice bath and then stirred for 1/2 h. The mixture was additionally stirred at ambient temperature for 2 h and then concentrated under reduced pressure to yield a yellow oil. The oil was neutralized with Et3N, and the amine salt was removed by extraction

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with CH3CN. The insoluble product was collected as white solid (1.16 g, 62.9%). 1H NMR (D2O): δ 4.00 (m, NH2CH(CH2OCH2CH2OCH3)C(O)OH, 1H), 3.90 (m, NH2CH(CH2OCH2CH2OCH3)C-(O)OH, 2H), 3.69-3.60 (m, NH2CH(CH2OCH2CH2OCH3)C(O)OH, 4H), 3.37 (s, NH2CH(CH2OCH2CH2OCH3)C(O)OH, 3H). 13C {1H} NMR (D2O): δ 172.52, 72.07, 70.79, 69.53, 59.08, 55.33. [R]D22 ) -8.31 (c ) 0.014, H2O). MALDITOF-MS, MH+: 163.17, calcd; 163.86, found. O-(2-(Methoxy)ethyl)-L-serine NCA. To O-(2-(Methoxy)ethyl)-L-serine (1.16 g, 7.11 mmol) were added THF (100 mL) and COCl2 (4.42 mL of a 1.93 M toluene solution), and the mixture was stirred for 5 h at room temperature. The resulting solution was concentrated to give a yellow oil as the crude product. The oil was crystallized from a tetrahydrofuran, toluene, and hexane mixture (1:4:4) at -30 °C to give the product as a white solid (1.01 g, 75.1%). FTIR (THF): 1859 cm-1 (νCO, s), 1792 cm-1 (νCO, s) 1H NMR(CDCl3): δ 7.50 (s, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH3, 1H), 4.78 (t, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH3,1H), 4.13, 3.97, 3.84 (m, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH3, 2H), 3.66 (s, RC(H)C(O)OC(O)NH, R ) -CH2OCH2CH2OCH3, 3H). 13C {1H} NMR(CDCl3): δ 168.65, 152.91, 71.78, 71.14, 69.25, 58.91, 58.80. [R]D23 ) -33.9 (c ) 0.054, THF). Poly(O-(2-(methoxy)ethyl)-L-serine), Poly(2). O-(2(Methoxy)ethyl)-L-serine NCA (1.01 g, 5.33 mmol) in THF (2 mL) was mixed with (PMe3)4Co (3.8 mg, 0.010 mmol) in THF (1 mL) and stirred for 18 h. The polymer was precipitated from this solution by addition to hexane (20 mL). The polymer was washed with H2O (5 mL), dialyzed to remove impurities, and then freeze-dried to give the product as a white solid (560 mg, 72%). FTIR (KBr): 1635 cm-1 (amide I, s br), 1521 cm-1 (amide II, s br). 1H NMR (LiBr (0.5 g/mL) in D2O): δ 8.68 (s, -(NHCH(CH2OCH2CH2OCH3)C(O))n-, 1H), 5.27 (s, -(NHCH(CH2OCH2CH2OCH3)C-(O))n-, 1H), 4.33-4.14 (m, -(NHCH(CH2OCH2CH2OCH3)C(O))n-, 6H), 3.86 (s, -(NHCH(CH2OCH2CH2OCH3)C(O))n-, 3H). 13C {1H} NMR (LiBr (0.5 g/mL) in D2O): δ 175.87, 72.48, 71.43, 70.81, 60.94, 54.72. [R]D23 ) -13.2 (c ) 0.068, LiBr (0.75 g/mL) in H2O). Molecular mass could not be determined since this polymer was insoluble in most solvents. (2-(2-Methoxyethoxy)ethyl)chloroformate. Di(ethylene glycol) monomethyl ether (5.0 g, 42 mmol) in THF (30 mL) was mixed with COCl2 (32.3 mL of a 1.93 M solution in toluene) at 0 °C and stirred for 1 h. It was then stirred at 10 °C for 2 additional hours. The solvent and excess phosgene were removed under reduced pressure to yield clear oil (7.0 g, 92%). FTIR (CH2Cl2): 1780 cm-1 (νCO, s). This compound was used without further purification. S-(2-(2-Methoxyethoxy)ethoxy)carbonyl-L-cysteine. LCysteine hydrochloride (2.00 g, 12.7 mmol) was dissolved in 1 N aqueous sodium bicarbonate (25.4 mL), and the solution was diluted with water (75 mL). The solution was cooled in an ice bath and then covered with ether (50 mL). (2-(2-Methoxyethoxy)ethyl)chloroformate (2.31 g, 12.7 mmol) was added to the solution in one portion with vigorous stirring for 1 h at 0 °C. The temperature was allowed to rise

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to 10 °C and stirred for an additional 2 h. The solvents were then removed under reduced pressure. The resulting solid was washed with methanol and the methanol layer was evaporated to yield the product as a white oil (2.21 g, 65%). FTIR (KBr): 1709 cm-1 (νCO, s). 1H NMR (D2O): δ 4.43 (m, NH2CH(CH2SC(O)OCH2CH2OCH2CH2OCH3)C(O)OH, 1H), 4.24 (m, NH2CH-(CH2SC(O)OCH2CH2OCH2CH2OCH3)C(O)OH, 2H), 3.83-3.47 (m, NH2CH(CH2SC(O)-OCH2CH2OCH2CH2OCH3)C(O)OH, 8H), 3.41 (s, NH2CH(CH2SC(O)OCH2CH2O-CH2CH2OCH3)C(O)OH, 3H). MALDITOF-MS, MH+: 303.76, calcd; 304.70, found. S-(2-(2-Methoxyethoxy)ethoxy)carbonyl-L-cysteine NCA. S-(2-(2-Methoxyethoxy)ethoxy)carbonyl-L-cysteine (2.21 g, 8.26 mmol) was dissolved in THF (100 mL), and COCl2 (5.13 mL of a 1.93 M solution in toluene) was then added. The mixture was stirred at ambient temperature for 5 h, and the solvent was then removed under reduced pressure to give a yellow oil. This oil was fractionally precipitated from THF and hexanes (2:4) to yield the product as a pale yellow oil (1.94 g, 80.1%). FTIR (THF): 1862 cm-1 (νCO, s), 1791 cm-1 (νCO, s), 1718 cm-1 (νCO, s). 1H NMR (CDCl3): δ 7.22 (s, RC(H)C(O)OC(O)NH, R ) -CH2SC(O)OCH2CH2OCH2CH2OCH3, 1H), 4.66 (t, RC(H)C(O)OC(O)NH, R ) -CH2SC(O)OCH2CH2OCH2CH2OCH3, 1H), 4.37 (m, RC(H)C(O)OC(O)NH, R ) -CH2SC(O)OCH2CH2OCH2CH2OCH3, 2H), 3.74-3.52 (m, RC(H)C(O)OC(O)NH, R ) -CH2SC(O)OCH2CH2OCH2CH2OCH3, 6H), 3.45-3.15 (m, RC(H)C(O)OC(O)NH, R ) -CH2SC(O)OCH2CH2OCH2CH2OCH3, 2H), 3.33 (s, RC(H)C(O)OC(O)NH, R ) -CH2SC(O)OCH2CH2OCH2CH2OCH3, 3H). 13C {1H} NMR (CDCl3): δ 169.45, 168.71, 151.93, 71.70, 70.39, 68.68, 68.00, 67.25, 58.96, 57.87. Poly(S-(2-(2-methoxyethoxy)ethoxy)carbonyl-L-cysteine), Poly(3). O-(2-(2-Methoxyethoxy)ethoxy)carbonyl-Lcysteine NCA (1.94 g, 6.61 mmol) was dissolved in THF (10 mL) and then mixed with (PMe3)4Co (3.8 mg, 0.010 mmol) in THF (1 mL). The initially homogeneous solution was stirred for 18 h to give the polymer as an off-white precipitate that was isolated by washing with diethyl ether (50 mL) (1.17 g, 70.9%). FTIR (KBr): 1635 cm-1 (amide I, s br), 1517 cm-1 (amide II, s br). 1H NMR (DMSO-d6): δ 8.35 (br s, -(NHCH(CH2SC(O)OCH2CH2OCH2CH2OCH3)C(O))n-, 1H), 4.51 (br s, -(NHCH-(CH2SC(O)OCH2CH2OCH2CH2OCH3)C(O))n-, 1H), 4.29 (br s, -(NHCH(CH2S-C(O)OCH2CH2OCH2CH2OCH3)C(O))n-, 2H), 3.613.33 (m, -(NHCH(CH2SC(O)OCH2-CH2OCH2CH2OCH3)C(O))n-, 8H), 3.24 (s, -(NHCH(CH2SC(O)OCH2CH2OCH2CH2O-CH3)C(O))n-, 3H). 13C {1H} NMR (DMSO-d6): δ 169.92, 169.20, 71.77, 69.64, 68.11, 66.78, 58.11, 40.39. Molecular mass could not be determined since this polymer was insoluble in most solvents. Results and Discussion. The synthesis of the EGmodified amino acids is shown in Scheme 1. The EG repeats were coupled onto the amino acids using ether and carbonate linkages, which provided both permanent and cleavable modifications, respectively. The modified amino acids were then converted to their corresponding amino acid-N-carboxyanhydride (NCA) monomers to allow subsequent polymerization (eq 1). The NCAs were polymerized using

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(PMe3)4Co initiator in THF (eq 2),12 whereupon the resulting

polymers were observed to precipitate during the course of the reactions. Polymer precipitation was undesirable since it prevented control of molecular weight by limiting the polymers to short chain lengths. Precipitation was likely caused by β-sheet formation with intermolecular H-bonding resulting in chain aggregation and phase separation. The NCA monomers were also polymerized in DMF, which is a good solvent for polypeptides. In these reactions, the polymers of 2 and 3 precipitated as before, while the polymer of 1 remained soluble up to degrees of polymerization ≈ 100. When KBr pellets of the polypeptides of 1-3 were examined by FTIR, they showed absorptions that were characteristic of β-sheet structures (amide I: 1635-1631 cm-1; amide II: 1523-1517 cm-1).13 Of these polymers, only poly(1) was found to be soluble in water. Circular dichroism (CD) analysis of this polymer in pH 7, deionized water revealed that it was in a “random coil” conformation (Figure 1).14 The CD spectra of this polymer were also invariant with solution pH and buffer strength, consistent with this result. However, the polymer was found to be aggregated by dynamic light scattering (DLS) measurements with an average aggregate diameter of 134 nm in pH 7, deionized water. These aggregates were likely associated through unorganized hydrogen bonds and could be completely dissociated in 3 M LiBr or 3 M guanidinium-HCl. Films cast from aqueous solutions of this polymer from a variety of buffers (3 < pH < 13, or in 10 mM TRIS (pH 7), phosphate (pH 7), or NaCl) all gave CD spectra indicative of the β-sheet conformation (Figure 1).14 Wide-angle X-ray scattering data from films of poly(1) revealed reflections that were also commensurate with the antiparallel β-sheet structure: 4.73 Å, H-bonding direction; 7.02 Å, chain axis; 16.3 Å, intersheet spacing.15 The large intersheet spacing reflects the packing of the long EG side chains between stacks of H-bonded sheets. With poly(2), this spacing was found to decrease to 11.9 Å due to the shorter side chains. Upon dissolution of poly(1) in water, solvent interactions with the EG side chains are apparently strong enough to disrupt the H-bonding found in the folded structure. This is reasonable since it is known that EG repeats interact strongly with water molecules through formation of ordered H-bonded structures,16 which can counterbalance the loss of amide H-bonding. It is likely that the association of water on the EG side chains increases their steric bulk to the extent that they repel one another and push the H-bonded β-strands apart. To probe the interaction of water with poly(1), CD spectra of this polymer were recorded a function of solvent composition. As solution composition was varied from pure

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Biomacromolecules, Vol. 2, No. 1, 2001 21

with methanol to assemble the β-strands. These features allow facile processing of β-sheet domains without use of strong denaturants or pH adjustments that may disrupt or precipitate other secondary structures present.

Figure 1. CD spectra of poly(1) (Mn ) 17 000): (A) in H2O ([poly(1)] ) 0.5 mg/mL, pH 7); (B) as a film cast from H2O.

Figure 2. CD spectra of poly(1) (Mn ) 17 000) in H2O/MeOH mixtures ([poly(1)] ) 0.5 mg/mL): (A) 100% H2O; (B) 50% H2O; (C) 25% H2O; (D) 10% H2O; (E) 5% H2O.

water to increasing percentages of methanol, a random conformation to β-sheet transformation was observed (Figure 2). This transition was also followed by DLS, which indicated that polymer particle size began to increase when the solution reached methanol compositions of 95% (v/v). A similar conformational change was also found to occur in a acetonitrile/water mixture (95/5 v/v). Aggregation of the polymer with increasing fractions of organic solvents, coincident with β-sheet formation, indicated that it was the strong interaction of poly(1) with water that destabilizes the β-sheet conformation. The solvent-dependent conformational properties of poly(1) provide a means for convenient processing of this polymer. Concentrated aqueous solutions can be cast into polypeptide films of high β-sheet content or can be treated

Acknowledgment. We thank Andrew P. Nowak and Professor Darrin J. Pochan for performing the X-ray measurements on the polymers. This work was supported by grants from the National Science Foundation under CAREER Award No. CHE-9701969, U.S. Army Research Office Multidisciplinary University Research Initiative, under Award No. DAAH04-96-1-0443, and partially supported by the MRSEC program of the National Science Foundation under Award No. DMR-9632716. T.J.D. is grateful for Alfred P. Sloan and Beckman Young Investigator fellowships. References and Notes (1) (a) Fasman, G. D. Prediction of Protein Structure and the Principles of Protein Conformation; Plenum Press: New York, 1989. (b) Bamford, C. H.; Elliot, A.; Hanby, W. E. Synthetic Polypeptides; Academic Press: New York, 1956. (2) Quadrifoglio, F.; Urry, D. W. J. Am. Chem. Soc. 1968, 90, 27602765. (3) (a) Berger, A.; Noguchi, J.; Katchalski, E. J. Am. Chem. Soc. 1956, 78, 4483-4488. (b) Ikeda, S.; Fasman, G. D. J. Mol. Biol. 1967, 30, 491-505. (c) Maeda, H.; Ikeda, S. Biopolymers 1971, 10, 16351648. (4) (a) Warwicker, J. O. Acta Crystallogr. 1954, 7, 565-573. (b) Marsh, R. E.; Corey, R. B.; Pauling, L. Biochim. Biophys. Acta 1955, 16, 1-34. (5) (a) Krejchi, M. T.; Atkins, E. D. T.; Waddon, A. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Science 1994, 265, 1427. (b) McGrath, K. P.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Am. Chem. Soc. 1992, 114, 727-733. (6) (a) Kaplan, D. L.; Fossey, S.; Mello, C. M.; Arcidiacono, S.; Senecal, K.; Muller, W.; Stockwell, B.; Beckwitt, R.; Viney, C.; Kerkam, K. MRS Bull. 1992, October, 41-47. (b) Liivak, O.; Blye, A.; Shah, N.; Jelinski, L. W. Macromolecules 1998, 31, 6733-6736. (7) Bohak, Z.; Katchalski, E. Biochemistry 1963, 2, 228-237. (8) Yu, M.; Nowak, A. P.; Pochan, D. P.; Deming, T. J. J. Am. Chem. Soc. 1999, 121, 12210-12211. (9) Zalipsky, S.; Lee, C. In Poly(EthyleneGlycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum: New York, 1992. (10) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, 1518-1520. (11) Klein, H.-F.; Karsh, H. H. Chem. Ber. 1975, 108, 944-955. (12) Deming, T. J. Macromolecules 1999, 32, 4500-4502. (13) Elliott, A. Proc. Royal Soc. A 1954, 221, 104-114. (14) (a) Adler, A. J.; Greenfield, N. J.; Fasman, G. D. Methods Enzymol. 1973, 27, 675-735. (b) Tiffany, M. L.; Krimm, S. Biopolymers 1969, 8, 347-359. (15) Elliott, A. Int. Congr. Biochem. (3rd Congr., Brussels) 1955, 106124. (16) (a) Bailey, F. E., Jr.; Callard, R. W. J. Appl. Polym. Sci. 1959, 1, 56-62. (b) Ataman, M. J. Macromol. Sci.sChem. 1987, A24, 967-976.

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