Surface-Initiated Vapor Polymerization of Various α-Amino Acids

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Surface-Initiated Vapor Polymerization of Various r-Amino Acids Nancy H. Lee and Curtis W. Frank* Department of Chemical Engineering, 381 North-South Mall, Stanford University, Stanford, California 94305-5025 Received May 8, 2002. In Final Form: October 15, 2002 Vapor deposition was used to synthesize surface-grafted polypeptides from a wide range of R-amino acids: γ-benzyl-L-glutamate, γ-methyl-L-glutamate, β-benzyl-L-aspartate, O-benzyl-L-serine, S-benzyl-Lcysteine, O-benzyl-L-tyrosine, L-tryptophan, L-phenylglycine, and L-phenylalanine. L-Alanine and L-valine were also examined, but surface grafting was not achieved. The thickness of the chemisorbed film, which ranged up to 800 Å, was measured using ellipsometry, and the conformation of the polymer, which can exist as an R-helix (right- or left-handed), β-sheet (parallel or antiparallel), and/or random coil, was determined by Fourier transform infrared spectroscopy (FTIR). Contact angle measurements were also performed to calculate the dispersive and polar surface energies of the film. The thermal stability of the monomer was important in the formation of the polymer film, and benzyl moieties present in the side chain seemed to provide that stability. The resulting film conformation on the surface corresponded well to that observed in the bulk. A large initial growth rate was observed for R-helical and parallel β-sheet structures, while a relatively slow rate was found for antiparallel β-sheets. The decrease in the rate of the polymerization at longer reaction times may be attributed to the physical blocking or chemical transformation of the amino end groups.

Introduction Proteins control biological activity by such methods as electron transfer, photoreactivity, and catalysis,1 and to take advantage of these material properties in the context of nonbiological applications, poly(amino acids) have been deposited onto various types of surfaces, which may prove to be important in such applications as biosensors,2 chiral separations,3-5 optical switches,6 and liquid crystal displays.7 Although homopolypeptides that are produced by synthetic means lack the complexity of naturally occurring proteins, these poly(amino acids) or polypeptides (we will use the terms interchangeably) exhibit the same secondary conformations as seen in proteins. There is potential for increased complexity of the polypeptides through copolymerization8 and for enhanced physical properties through the incorporation of synthetic amino acids.9 Chemical bonding of poly(amino acids) to a substrate can be accomplished by modifying the surface with initiators, which initiate the polymer synthesis (graftingfrom approach).10 Many research groups have used this approach to synthesize poly(amino acids) in solution on amine-modified gold or silicon substrates and to examine the film growth rate, the effect of monomer and initiator * To whom correspondence should be addressed. Telephone (650) 723-4573. Fax: (650) 723-9780. E-mail: curt@ chemeng.stanford.edu. (1) Sanda, F.; Endo, T. Macromol. Chem. Phys. 1999, 200, 26512661. (2) Deming, T. J. Adv. Mater. 1997, 9, 299-311. (3) Aoki, T. Prog. Polym. Sci. 1999, 24, 951-993. (4) Lee, N. H.; Frank, C. W. Submitted for publication in Langmuir. (5) Lee, N. H.; Frank, C. W. Polymer 2002, 43, 6255-6262. (6) Whitesell, J. K.; Chang, H. K. Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. A 1994, 240, 251-258. (7) Machida, S.; Urano, T. I.; Sano, K.; Kato, T. Langmuir 1997, 13, 576-580. (8) Wieringa, R. H.; Siesling, E. A.; Werkman, P. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17, 6491-6495. (9) Tang, Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, W. A.; DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790-2796. (10) Chang, Y. C.; Frank, C. W. Langmuir 1996, 12, 5824-5829.

concentrations, and the orientation of the helix resulting from the polymerization.11-14 Surface polymerization can also be performed in the melt by spin-coating an N-carboxy anhydride (NCA) monomer over a silicon substrate modified with amino end groups and then heating the sample above the melting point of the monomer, producing poly(γ-methyl-L-glutamate) films with R-helix and β-sheet conformations.15 As a complement to solution and melt polymerizations, Chang and Frank16 developed a novel method of surface synthesis, known as NCA vapor deposition polymerization, in which the monomer in the vapor phase reacts with the surface-attached initiator to grow the polypeptide film. Poly(γ-benzyl-L-glutamate) and poly(γ-methyl-L-glutamate) were synthesized using this method, and the thickness was varied by changing the duration and temperature of the reaction as well as the distance of the substrate from the NCA monomer. The advantage of the vapor deposition method is that it is a solventless procedure; moisture and impurities from the solvent are eliminated, thus minimizing the oligomerization of the monomer.17 The polymerization of amino acid monomers in a vacuum and at elevated temperatures is not a new concept, as it has been performed since the 1950s. In a series of experiments by Berger and Sela,18-21 bulk polymerization was carried out for β-benzyl-L-aspartate, S-benzyl-L(11) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73-76. (12) Heise, A.; Menzel, H.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M. Langmuir 1997, 13, 723-728. (13) Wieringa, R. H.; Siesling, E. A.; Geurts, P. F. M.; Werkman, P. J.; Vorenkamp, E. J.; Erb, V.; Stamm, M.; Schouten, A. J. Langmuir 2001, 17, 6477-6484. (14) Wieringa, R. H.; Siesling, E. A.; Werkman, P. J.; Angerman, H. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17, 6485-6490. (15) Wieringa, R. H.; Schouten, A. J. Macromolecules 1996, 29, 30323034. (16) Chang, Y. C.; Frank, C. W. Langmuir 1998, 14, 326-334. (17) Kricheldorf, H. R. R-Amino Acid-N-Carboxy-Anhydrides and Related Heterocycles, 1st ed.; Springer-Verlag: Berlin, 1987. (18) Frankel, M.; Berger, A. J. Org. Chem. 1951, 16, 1513-1518. (19) Berger, A.; Noguchi, J.; Katchalski, E. J. Am. Chem. Soc. 1956, 78, 4483-4488.

10.1021/la020432i CCC: $25.00 © 2003 American Chemical Society Published on Web 01/17/2003

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Table 1. Properties and Deposition Conditions (Time and Temperature) of the Poly(amino acids) melting point of NCA (°C)

deposition time (h)

deposition temp (°C)

solventa DCA/chloroform DCA/chloroform DMF

γ-benzyl-L-glutamate γ-methyl-L-glutamate β-benzyl-L-aspartate

91-93 94-98 126-129

Class I: Esters 2 2 3

105 110 140

O-benzyl-L-serine S-benzyl-L-cysteine

71-73 105-106

Class II: Heteroatom (Cβ) 2 2.5

80 90

O-benzyl-L-tyrosine L-tryptophan L-phenylglycine L-phenylalanine

141 144-146 124-129 88-89

Class III: Aromatics 2.3 4.6 2 2

140 140 135 100

L-alanine

90-92 68

Class IV: Aliphatics 2 2

100 80

L-valine

DCA DCA DMF DMF DMSO DCA

a Solvent used to remove physisorbed oligopeptides after vapor polymerization. DCA, dichloroacetic acid; DCA/chloroform, 10 vol % DCA and 90 vol % chloroform; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide.

cysteine, L-tryptophan, and L-phenylalanine at temperatures of 110-150 °C and at pressures from 10-5 to 10-3 mm. This shows that, even without any initiator, these monomers are able to polymerize thermally at the given conditions. In fact, thermal polymerization can contribute a substantial amount to the overall NCA reaction of the present work. In our study, we will demonstrate the applicability of the vapor deposition polymerization process for synthesizing a wide range of polypeptides. Chang and Frank16 only studied two polypeptides using this process, so we chose synthetic and naturally occurring amino acids based on the characteristic structure of the side chain and on the ease of synthesizing the respective NCA monomers. Our main objective was to better understand the formation and growth of grafted polypeptide films by vapor deposition. The resulting films were characterized by ellipsometry, Fourier transform infrared spectroscopy (FTIR), and contact angle, and the conformation and growth rates of the polymers were compared to those observed in the bulk. Experimental Section Materials. The amino acids, triphosgene, and (γ-aminopropyl)triethoxysilane (APS) were purchased from Sigma-Aldrich. Tetrahydrofuran (THF) and hexane were distilled over sodium. Anhydrous petroleum ether and ethyl acetate were used as purchased. NCA Synthesis. The NCA monomers were synthesized according to the method outlined by Daly and Poche22 and purified by recrystallization (O-benzyl-L-tyrosine, L-alanine, and L-valine) or by rephosgenation (γ-benzyl-L-glutamate, γ-methyl-L-glutamate, β-benzyl-L-aspartate, S-benzyl-L-cysteine, and L-phenylalanine).23 For O-benzyl-L-serine and L-phenylglycine, after the initial reaction with triphosgene, the solvent was removed by a rotary evaporator, leaving a yellowish oil. Petroleum ether was added to precipitate the monomer, and it was purified by recrystallization from ethyl acetate and petroleum ether to obtain the final product. When triphosgene was added to the Ltryptophan suspension, a clear solution was instantly formed. The solution then turned to a clear lime green color and finally to a cloudy green mixture. After adding hexane and placing the flask in the freezer overnight, a yellow cake and white needlelike crystals were formed. The yellow cake was removed and the white needles were recrystallized using THF and hexane. (20) Patchornik, A.; Sela, M.; Katchalski, E. J. Am. Chem. Soc. 1954, 76, 299-300. (21) Sela, M.; Berger, A. J. Am. Chem. Soc. 1955, 77, 1893-1898. (22) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859-5862. (23) Dorman, L. C.; Shiang, W. R.; Meyers, P. A. Synth. Commun. 1992, 22, 3257-3262.

The yields ranged from 10 to 50%, which were lower than literature values (50-90%).22,23 The structures of the NCA monomers were verified by 1H NMR,17,22 and the melting points are given in Table 1. Substrate Preparation. To prepare the substrate for polymerization, a surface modification procedure, adapted from Chang and Frank,16 was used. A piece of double-polished silicon (100) (1.5 cm × 2.5 cm) from Silrec Corp. was cleaned using a Piranha solution (30% hydrogen peroxide and concentrated sulfuric acid in 30:70 volume ratio). (Piranha solution is very reactive; use extreme caution when working with this solution.24) The wafer was placed in a beaker containing this solution, and the beaker was sonicated for 45 min to remove hydrocarbon impurities. The substrate was then rinsed with Millipore water and ethanol and dried using nitrogen (N2). To deposit the initiator on the surface, we placed the substrate in a Schlenk tube with 0.03 wt % APS in acetone for 1 h at 50 °C. The wafer was removed and rinsed with acetone and chloroform and dried using N2. The substrate was used immediately for vapor deposition. Vapor Deposition. The NCA monomer (0.01 g) was spread at the bottom of a glass reactor. The APS-modified substrate was supported on a glass holder and placed 1.5 cm above the monomer. The chamber was evacuated with a rough pump and then backfilled with N2 for two cycles. Afterward, the system was heated to approximately 10 °C above the melting point of the polymer for 2 h. During the course of the polymerization, the chamber was continuously evacuated using a turbo pump (5 × 10-5 mbar) to remove carbon dioxide that evolved from the polymerization, thus driving the reaction. The maximum temperature for the reaction was limited to 140 °C due to the thermal degradation of the O-ring in the valve that connects the reactor to the pump. However, temperatures up to 200 °C can be reached if the O-ring is replaced with Viton (fluorocarbon) or silicone O-rings. To remove any physisorbed oligopeptides adsorbed on the surface, we soaked the substrates overnight in a good solvent for the polymer, such as chloroform, dichloroacetic acid (DCA), and dimethyl sulfoxide. The substrates were rinsed with the same solvent and dried over N2. The reaction temperatures and the solvents used to remove the physisorbed materials are also listed in Table 1. The polymer deposition occurred on both sides of the silicon substratesthe side facing toward and that facing away from the monomer in the reaction vessel. The side facing the NCA monomer was used for all thickness measurements except in the case of poly(γ-benzyl-L-glutamate). The side facing away from the γ-benzyl-L-glutamate monomer was used for the measurements, since it produced a more uniform film with the same thickness. Ellipsometry. An ellipsometer from Gaertner Scientific Corp. (model L116C) with a He-Ne laser was used to measure film thickness. The laser angle of incidence was set at 70°, and a (24) Dobbs, D. A.; Bergman, R. G.; Theopold, K. H. Chem. Eng. News 1990, 68, 2-2.

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refractive index of 1.46 was used for all the measurements. The reported film thickness was averaged from at least five different spots on each sample. FTIR. A Perkin-Elmer Spectrum 2000 FTIR was used to determine the conformation of the deposited film. The spectrum was taken in transmission mode, and a mercury cadmium telluride N2 cooled detector was used. For each sample, 1064 scans were taken at a resolution of 4 cm-1. The resolution limit for the FTIR was approximately 30 Å; below this film thickness, it was difficult to detect the polymer conformation with this technique. Contact Angle. Contact angle measurements were performed using the FTA 200 dynamic contact angle system from Camtel Ltd. The film surfaces were first cleaned by rinsing with chloroform. The advancing and receding contact angles of water (surface tension (γLV) ) 72.8 mN/m) and diiodomethane (γLV ) 50.8 mN/m) were measured by recording the contact angle of a drop on the surface as the solvent was pumped into and withdrawn from the drop at 0.1 µL/s. These measurements were performed on two locations on the sample, due to the size limitation of the sample; however, these values were consistent from sample to sample. The dispersive (γLD) and polar (γLP) components of the surface tension were 21.8 and 51.0 mN/m, respectively, for water and 49.5 and 1.3 mN/m for diiodomethane.25

Results and Discussion Vapor Deposition. Although many classification systems already exist for naturally occurring amino acids,26-28 we utilized a system developed by Koolman and Ro¨hm28 because it was more descriptive of the intermolecular forces involved. Categories were also added to accommodate the synthetic amino acids that we included in this study. As shown in Figure 1, we divided the amino acids under study into four different classes. Class I consists of molecules with an ester functional group; this includes the L-glutamates (γ-benzyl and γ-methyl) and β-benzyl-L-aspartate. A heteroatom (oxygen or sulfur) is located next to the β-carbon for the compounds in class II. The aromatic poly(amino acids) that did not fit into the first two classes were grouped into class III, which contains the monomers O-benzyl-L-tyrosine, L-tryptophan, L-phenylglycine, and L-phenylalanine. Class IV is composed of L-alanine and L-valine, which contain aliphatic side chains. The NCA monomers undergo a ring-opening polymerization in the presence of amino groups to form the polymer chain, as depicted in Figure 2, and the vapor deposition was performed with the various classes of monomers. The initial film thickness, containing both chemisorbed and physisorbed polypeptides, was approximately two to three times larger than the film thickness measured after removal of the physisorbed material. The ellipsometric thicknesses for the chemisorbed film are presented in Table 2. No polymer films were present for class IV (aliphatics) poly(amino acids). This may be attributed to the thermal instability of these aliphatic monomers. The vapor deposition reaction is carried out at low pressures and at elevated temperatures to ensure that the monomers will vaporize and react with the initiator at the surface. This process requires that the monomers be thermally stable. The NCA of alanine is a highly reactive monomer. Although the NCA of valine is somewhat more stable, both monomers are able to undergo solid thermal polymerization at temperatures below 0 °C.17 Therefore, these (25) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 17411747. (26) Devlin, T. M. Textbook of Biochemistry: With Clinical Correlations, 3rd ed.; Wiley-Liss Inc.: New York, 1992. (27) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Co.: New York, 1995. (28) Koolman, J.; Ro¨hm, K.-H. Color Atlas of Biochemistry; Thieme: Stuttgart, Germany, 1996.

Figure 1. Structures and classes of poly(amino acids) studied for surface polymerization using vapor deposition.

Figure 2. Surface synthesis reaction of the polypeptide film. The monomer undergoes a ring-opening polymerization, initiated by (γ-aminopropyl)triethoxysilane (APS) molecules that are bonded to the substrate. The products of the reaction are the polypeptide film and carbon dioxide.

NCA monomers were stored under N2 at -20 °C, and polymerizations were performed within 2 weeks of NCA synthesis. When these monomers are in a molten state, the polymerization is fast and may only require a few

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Table 2. Film Thickness and Conformation of Deposited Polypeptides % R-helix film thickness (Å) γ-benzyl-L-glutamate γ-methyl-L-glutamate β-benzyl-L-aspartate

580 ( 40 110 ( 5 310 ( 30

O-benzyl-L-serine S-benzyl-L-cysteine

80 ( 10 130 ( 5

O-benzyl-L-tyrosine L-tryptophan L-phenylglycine L-phenylalanine

800 ( 110 220 ( 10 50 ( 5 80 ( 10

right-handed

% β-sheet

left-handed

parallel

Class I: Esters 100 98

antiparallel

% random

2 85

15

Class II: Heteroatom (Cβ) 62 53

38 47

28 25

38

Class III: Aromatics 100 100 72 37 Class IV: Aliphatics

L-alanine L-valine

no film present no film present

minutes for completion,17 and the thermal polymerization may be completed before vaporization is able to occur. Even though no films were synthesized for class IV peptides, films of relatively large thickness were formed from NCA monomers of class I (esters). The film thicknesses for class II (heteroatom at Cβ) were smaller, ranging from 80 to 130 Å. For class III (aromatics), O-benzyl-Ltyrosine and L-tryptophan produced films greater than 200 Å thick, while film thicknesses for L-phenylglycine and L-phenylalanine were relatively low (50-80 Å). The aromatic side group in these monomers may provide the thermal stability necessary for vapor deposition to occur. This proposal is supported by O-benzyl-L-tyrosine, which has two aromatic groups in its side chain; it produced the thickest polypeptide film (800 Å). For these polymers, it is possible that thermal polymerization or decomposition may be occurring before and during the actual deposition process. However, the thermal polymerization may be slow, thereby still allowing vaporization to occur. Conformation. FTIR was used to probe the conformation of the polypeptides grafted onto the substrate. Determining the conformation from infrared spectra has been well-studied, and the observed peak locations are summarized in Table 3.29-35 The shifts in the location of the peaks of the amide bonds are due to the vibrational interactions between the peptide groups and the different forms of hydrogen bonding among the amino acid residues.30 As an example, both R-helices and β-sheets have regular hydrogen bonding; however, there is a difference in the pattern of the hydrogen bonding and in the geometric orientation of the amide bonds. This is responsible for the shifts in the amide I (backbone CdO stretch), which occurs in the region of 1600-1700 cm-1;34 the amide I peak is located at higher wavenumbers for a left-handed R-helix (1668 cm-1) compared to a right-handed R-helix (1655 cm-1).32,33 The strong band of the parallel β-sheet (adjacent chains oriented in the same direction) occurs approximately at 1636-1640 cm-1, while the location for an antiparallel β-sheet (adjacent chains oriented in the (29) Blout, E. R.; Loze, C. D.; Bloom, S. M.; Fasman, G. D. J. Am. Chem. Soc. 1960, 82, 3787-3789. (30) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712719. (31) Lal, B. B.; Nafie, L. A. Biopolymers 1982, 21, 2161-2183. (32) Sen, A. C.; Keiderling, T. A. Biopolymers 1984, 23, 1533-1545. (33) Sen, A. C.; Keiderling, T. A. Biopolymers 1984, 23, 1519-1532. (34) Singh, B. R. Basic Aspects of the Technique and Applications of Infrared Spectroscopy of Peptides and Proteins; Singh, B. R., Ed.; American Chemical Society: Washington, DC, 2000; pp 2-37. (35) Bonora, G. M.; Moretto, V.; Toniolo, C.; Anzinger, H.; Mutter, M. Int. J. Pept. Protein Res. 1983, 21, 336-343.

Table 3. Infrared Spectral Peaks of the Various Conformations of Polypeptides (Units Given in cm-1)b

a s, strong bond; w, weak bond. b The schematic representation of these conformations on the surface is also given, with the hydrogen bonding indicated by the dotted line.

opposite direction) is between 1622 and 1632 cm-1.30,35 The amide II (C-N stretch and N-H deformation) region is present at 1500-1600 cm-1, but this region also includes the contribution from the aromatic ring. Therefore, it is not as sensitive to the changes in conformation compared to the amide I band.34,35 The FTIR spectra are shown in Figure 3. The percentage of R-helical (right- or left-handed), β-sheet (parallel or antiparallel), and random conformations, shown in Table 2, was calculated by deconvolution of the amide I region (1620-1670 cm-1) using a Lorentzian fit (Origin software) and then taking the ratio of the peak areas. For class I (esters), the presence of a peak at 1735 cm-1 is due to the side chain CdO carbonyl stretch from the ester functional group. The thermodynamically stable conformation of poly(γ-benzyl-L-glutamate) (class I) is an R-helix for degree of polymerization (DP) > 10 and β-sheet for DP < 10 and DP > 4.17,36 For methyl glutamates, however, the helix is less stable and both right-handed R-helix and antiparallel β-sheet conformations are interchangeable, depending on the solvent. For example, R-helical conformations have been observed in chloroform or N,N-dimethylformamide (DMF), while β-sheet predominates in formic acid or pyridine.37,38 We have found that at the given thicknesses, γ-benzyl- and γ-methyl-L-glutamates were in the helical conformation. Poly(β-benzyl-L-aspartate) forms lefthanded R-helices in chloroform with a small amount of DCA and converts to a random coil as the concentration (36) Blout, E. R.; Asadourian, A. J. Am. Chem. Soc. 1956, 78, 955. (37) Chang, Y.-C., Ph.D. Thesis, Stanford, 1998. (38) Gillgren, H.; Stenstam, A.; Ardhammar, M.; Norden, B.; Sparr, E.; Ulvenlund, S. Langmuir 2002, 18, 462-469.

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Figure 3. FTIR spectra of the polypeptide films.

of DCA increases.39 In the solid state, right- and lefthanded R-helices, left-handed 410 ω-helices (amide I ) 1675 cm-1, amide II ) 1536 cm-1), and β-sheet conformations have all been observed for this polymer depending on temperature.40,41 In the vapor-deposited films, poly(β-benzyl-L-aspartate) existed as a left-handed R-helix with parallel β-sheet components. Class II compounds contain a heteroatom (oxygen or sulfur) next to the β-carbon in the side chain. Only β-sheet and random structures are supported for these polymers, and the antiparallel β-sheet is the thermodynamically stable conformation.29,42 This can be explained by the dipole-dipole interaction between the heteroatom and the adjacent peptide group, which prevents the formation of intramolecular hydrogen bonds and destabilizes the helix. For the vapor-deposited films, both the random and (39) Hayashi, Y.; Teramoto, A.; Kawahara, K.; Fujita, H. Biopolymers 1969, 8, 403-420. (40) Akieda, T.; Mimura, H.; Kuroki, S.; Kurosu, H.; Ando, I. Macromolecules 1992, 25, 5794-5797. (41) Riou, S. A.; Hsu, S. L.; Stidham, H. D. Langmuir 1998, 14, 30623066. (42) Toniolo, C.; Bonora, G. M.; Palumbo, M.; Peggion, E.; Stevens, E. S. Biopolymers 1978, 17, 1713-1727.

antiparallel β-sheets were observed for O-benzyl-L-serine and S-benzyl-L-cysteine. For class III polymers, parallel β-structure was present in the O-benzyl-L-tyrosine film. In a study by Bonora et al.,35 oligopeptides of O-benzyl-L-tyrosine existed predominantly in the parallel β-sheet structure for DP > 4; likewise, our resulting film was in the same conformation. The conformation of poly(L-tryptophan) in DMF is R-helical and in dimethyl sulfoxide is random.19 We have found that films of L-tryptophan were in the R-helical conformation. L-Phenylglycine and L-phenylalanine films adopted right-handed R-helical and antiparallel β-sheet conformations. In the solid state, polymers of phenylglycine have been found to be in the helical and antiparallel β-sheet conformations,43,44 and polymers of phenylalanine in helical and both antiparallel and parallel β-sheet conformations.42,45,46 (43) Toniolo, C.; Crisma, M.; Formaggio, F.; Polese, A.; Doi, M.; Ishida, T.; Mossel, E.; Broxterman, Q.; Kamphuis, J. J. Mol. Struct. 1988, 172, 395-400. (44) Palumbo, M.; Cosani, A.; Terbojevich, M.; Peggion, E. Int. J. Biol. Macromol. 1981, 3, 91-96. (45) Peggion, E.; Strasorier, L.; Cosani, A. J. Am. Chem. Soc. 1970, 92, 381-386.

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Table 4. Contact Angle and the Dispersive (γsD), Polar (γsP), and Total (γs) Surface Energies of Polypeptide Films contact angle (deg) water

diiodomethane

advancing

receding

advancing

γ-benzyl-L-glutamate γ-methyl-L-glutamate β-benzyl-L-aspartate

85 74 85

82 71 82

Class I: Esters 42 45 41

O-benzyl-L-serine S-benzyl-L-cysteine

86 83

82 78

Class II: Heteroatom (Cβ) 42 48

O-benzyl-L-tyrosine L-tryptophan L-phenylglycine L-phenylalanine

85 84 88 85

82 79 84 82

Class III: Aromatics 43 13 42 44

Although each polypeptide has a thermodynamically stable conformation based on the nature of the side chain and molecular weight (MW), some general statements can be made relating film thickness to conformation. Polymers predominantly in the helical or parallel β-sheet conformation produced relatively thick films, but relatively thin films were observed for poly(amino acids) having both antiparallel β-sheet and random coil conformations. Low conversions have also been seen for antiparallel β-sheets in solution because as the chain grows, the strand folds back to form an interchain antiparallel sheet. Consequently, the chain ends become deactivated due to steric reasons and a “physical death” of the polymer occurs.17 This may explain the small film thickness observed for polymers of O-benzyl-L-serine, S-benzyl-L-cysteine, Lphenylglycine, and L-phenylalanine, in which 30-60% of the film was in the β-sheet conformation. The conformations of the synthesized polymer films are consistent with those found in the bulk or in solution. This indicates that although the polymer is constrained to the surface, it has the flexibility to interact with its own or an adjacent chain to form hydrogen bonds and to adopt the various secondary structures. The vapor deposition method provides the ability to create surfaces with a range of secondary conformations. Contact Angle. The advancing and receding water and diiodomethane contact angles are presented in Table 4. The surfaces of the polypeptide films were relatively hydrophobic, with advancing water contact angles ranging from 74 to 88°. Poly(γ-methyl-L-glutamate), which was the only nonaromatic polypeptide to form a film, had the lowest water contact angle; advancing contact angles for all other films were greater than 80°. The advancing diiodomethane contact angles ranged from 13 to 48°. The receding contact angle of diiodomethane could not be measured for poly(L-tryptophan) because the solvent wet the film. The total surface free energy of the solid film (γS), which is a sum of the dispersive (γSD) and polar components (γSP), can be determined by using the following equation, which relates the interaction of the liquid and the surface to the surface energies:25

( )

1 + cos θ ) 2xγSD

xγLD γLV

( )

+ 2xγSP

x γLP γLV

surface energy (mN/m)

receding

γsD

γsP

γs

41 40 37

36 34 38

3 8 3

39 42 41

39 44

37 33

3 5

40 38

40

36 48 37 36

3 2 2 3

39 50 39 39

41 42

of water and diiodomethane into eq 1, are also listed in Table 4. The γSD of the polypeptide films (33-48 mN/m) was significantly larger than the γSP (2-8 mN/m). The contact angles and the surface energies are governed by the conformation and orientation of the polypeptide and the nature of the side chain, which determines the packing density in the secondary structure.12,47-49 In general, dispersive forces are greater for helical conformations due to nonpolar side groups that radially distribute around the helix backbone.49 In β-sheets, polar force contributions are greater if the side chains pack loosely within the sheet, allowing the solvent to penetrate the sheet and hydrogen bond with the chain.48,49 Although definitive conclusions on the relationship between conformation and surface energies could not be made in our study, some trends were observed. The dispersive surface energies of the helical poly(amino acids) γ-benzyl-Lglutamate and L-tryptophan were slightly larger than the β-sheet forming polymers S-benzyl-L-cysteine and Obenzyl-L-tyrosine. The structure of the side chain may also play a role in determining the surface energies. For example, poly(γ-methyl-L-glutamate), which lacks an aromatic group and contains two oxygens in its side chain, had a relatively high polar surface energy (8 mN/m), while poly(O-benzyl-L-tyrosine) with two aromatic groups had a relatively large dispersive surface energy (36 mN/m). When comparing the total surface energies, the surface energy of vapor-deposited poly(γ-benzyl-L-glutamate) (39 mN/m) and poly(γ-methyl-L-glutamate) (42 mN/m) were similar to the values obtained by casting these polymers from solution (36 and 41 mN/m, respectively).49 Kinetic Study. The kinetics of the vapor deposition process was examined by varying the reaction time but keeping the temperature fixed at the same value as for the earlier static experiments; the resulting film thickness and conformation were measured. These experiments were carried out for γ-benzyl-L-glutamate and β-benzyl-Laspartate from class I and O-benzyl-L-tyrosine, L-tryptophan, and L-phenylalanine from class III because they had either formed relatively thick films or showed interesting secondary structures. Figure 4 gives the ellipsometric thickness as a function of reaction time, which was fit using a transient Langmuir isotherm model.50,51 The initial growth rates, calculated

(1)

The γSD and γSP of each surface, calculated by inserting the average of the advancing and receding contact angles (46) Miyamoto, H.; Takezaki, R.; Komoto, T.; Ando, I.; Saito, H.; Ozaki, T.; Shoji, A. J. Mol. Struct. 1988, 172, 395-400.

(47) Enriquez, E. P.; Gray, K. H.; Guarisco, V. F.; Linton, R. W.; Mar, K. D.; Samulski, E. T. J. Vac. Sci. Technol., A 1992, 10, 2775-2782. (48) Baier, R. E. Macromolecules 1970, 3, 70-79. (49) Shibata, A.; Tsukamoto, R.; Ueoka, T.; Ueno, S.; Yamashita, T. Colloids Surf., A 1993, 78, 189-192. (50) Fogler, H. S. Elements of Chemical Reaction Engineering, 2nd ed.; P T R Prentice-Hall: Englewood Cliffs, NJ, 1992. (51) Chen, S. H.; Frank, C. W. Langmuir 1989, 5, 978-987.

Vapor Polymerization of Various R-Amino Acids

Langmuir, Vol. 19, No. 4, 2003 1301

Table 5. Conformation of the Polypeptides as a Function of Reaction Time and Thicknessa % R-helix time (h) γ-benzyl-L-glutam ate

β-benzyl-L-aspart ate

O-benzyl-L-tyrosine

L-tryptophan

L-phenylalanine

a

0.5 1 2 4 0.25 0.5 1 3 0.5 1 1.5 2.3 3 0.5 1 2 4.6 0.5 1 2 2.8

thickness (Å) 240 ( 10 320 ( 50 580 ( 40 580 ( 60 50 ( 5 240 ( 30 260 ( 10 310 ( 30 140 ( 20 200 ( 10 410 ( 30 800 ( 110 610 ( 50 30 ( 5 110 ( 10 170 ( 10 220 ( 10 40 ( 5 50 ( 5 80 ( 10 70 ( 10

right-handed

% β-sheet

left-handed

parallel

antiparallel

% random

growth rate (Å/h)

Class I: Esters 100 100 100 100

320

55 67 85

100 45 33

300

15

Class III: Aromatics 100 100 100 100 100 no film 100 100 100 28 32 37 53

290

100 33 30 25 14

38 38 38 33

60

The initial growth rates are also given.

Figure 4. Film thickness of (a) class I (esters) and (b) class III (aromatics) poly(amino acids) plotted as a function of reaction time.

from the slope of the linear region, are listed in Table 5. When the growth rates of vapor-deposited polymers are compared to polymerization in solution, complications

arise because NCA polymerizations are affected by many factors, including the nature of the initiator (primary, secondary, or tertiary amines), the nature of the side chain, the solvent polarity, temperature, and the monomer and initiator concentrations.17 The growth rates for the polymerization in the solution were compared for poly(γbenzyl-L-glutamate) since kinetic data for this compound were available.13,52 In solution polymerization of γ-benzyl-L-glutamate, two different rates of propagation were observed by Lundberg and Doty.52 The first rate constant was linked to the formation of β-sheet structures. The second rate constant, which was five times larger, occurred when the chain had an average of eight residues, and this fast propagation was due to the helical growth of the poly(amino acid). For polymerization in dioxane initiated by n-hexylamine with monomer-to-initiator ratio of 20, a rate of 0.8 and 4 DP/h was estimated for the slow and fast propagations, respectively. A linear growth followed by saturation was found when the monomer-to-initiator ratio was increased to 300. In the case of vapor polymerization, the initial growth rate of γ-benzyl-L-glutamates was approximately 320 Å/h. The helix orientation of this polypeptide by surfaceinitiated polymerization in solution is 32° with respect to the substrate.14 Therefore, the chain growth rate can be extrapolated to 600 Å/h or 400 DP/h because one residue contributes a 1.5 Å rise along the helical axis. Comparing the helical growth rates, the vapor polymerization rate was approximately 100 times larger than solution polymerization. Wieringa et al.13 observed a similar trend of linear growth followed by relatively constant film thickness for the solution surface polymerization of γ-benzyl- and γ-methyl-L-glutamates on silicon substrates. In their work, the linear regime occurred during the first 5-6 h of polymerization, producing 400 Å poly(γ-benzyl-Lglutamate) films; therefore, the film growth rate can be approximated to be 80 Å/h or 100 DP/h. Although the (52) Lundberg, R. D.; Doty, P. J. Am. Chem. Soc. 1957, 79, 39613972.

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Langmuir, Vol. 19, No. 4, 2003

surface solution polymerization is about 25 times faster than solution polymerization, this rate is still approximately four times slower than for vapor deposition of γ-benzyl-L-glutamate. Relatively large growth rates (approximately 300 Å/h) were found for class I esters (γ-benzyl-L-glutamate and β-benzyl-L-aspartate), and this rapid propagation may be due to the helical growth of the polymer. However, the rate for poly(L-tryptophan) (100 Å/h), which also existed as an R-helix, was slower. The melting point for the NCA monomer of L-tryptophan was 144-146 °C, but the maximum deposition temperature was limited to 140 °C by equipment restriction. The vaporization rate of the monomer may be decreased by the low reaction temperature, causing a decrease in the growth rate for Ltryptophan. A large growth rate (290 Å/h) was observed for the formation of parallel β-sheets (O-benzyl-L-tryrosine). The rate of propagation for antiparallel β-sheet conformation in solution is much slower than the rate for helical growth due to the steric hindrance caused by the folding back of the strands, preventing monomers from diffusing to the growing chain.17 This is consistent with the slow growth rate observed for L-phenylalanine (60 Å/h), in which 30% of the film was in the antiparallel β-sheet conformation. The poly(amino acids) γ-benzyl-L-glutamate, O-benzylL-tyrosine, and L-tryptophan showed no variation in conformation for the thicknesses that were probed (Table 5). However, β-benzyl-L-aspartate and L-phenylalanine showed a similar trend; as thickness increased, the percentage of the R-helical conformation increased. For these compounds, the helix is the thermodynamically stable conformation,39,42,45 and the helix content increased as the molecular weight of the polymer increased. Transient Langmuir Kinetic Model. Although the Langmuir kinetic equation is generally used to describe the adsorption of a gas or vapor onto active sites in catalytic processes,50,53 this equation has also been applied in the physiochemisorption of self-assembled monolayers.51,54 In a study by Chen and Frank,51 the adsorption of alkanoic acid on alumina could be fitted using a transient Langmuir adsorption model when the fatty acids were longer than C12. In addition, both the physical and chemical adsorption of cyclic polymethylsiloxane onto alumina were investigated by Cosgrove et al.,54 who found that the irreversible chemisorption was preceded by physisorption. Since the film growth rate for our vapor deposition was also modeled using a Langmuir adsorption isotherm, we can consider the amino end groups initially present on the substrate as “active sites” on a surface. As polymerization progresses, the amino end groups required for the propagation of the chain become chemically modified through the formation of hydantoic acid end groups or through cyclization.21,52 The decrease in the reaction rate with time can also be attributed to the physical blocking of the amino end groups by physisorbed materials, which accounted for 50-70% of the initial film thickness. These compounds, synthesized by thermal polymerization, adhere to the surface during the deposition reaction. They may block the ends of the growing chemisorbed polymers, preventing the monomers from diffusion to the propagating chain ends. Because of the chemical modification or physical blockage, the number of active sites available for chain propagation decreases. Therefore, the overall po(53) Adamson, A. W.; Gast, A. P. Physical Chemistry of Surfaces, 6th ed.; John Wiley & Sons: New York, 1997. (54) Cosgrove, T.; Prestidge, C. A.; Vincent, B. J. Chem. Soc., Faraday Trans. 1990, 86, 1377-1382.

Lee and Frank

lymerization rate decreases, and the film thickness eventually plateaus. Molecular Weight. The MW of the vapor-deposited polypeptides was not determined explicitly; however, estimates can be made by examining the orientation of the helix and the β-sheet dimensions. We have found that the MW of γ-benzyl-L-glutamate from thermal polymerization at 105 °C and 5 × 10-5 mbar for 3 h was approximately 2000, determined from viscometry. The helix orientation for γ-benzyl-L-glutamate is 32° from the substrate.14 For a 580 Å film, the corresponding axial length of the helix is approximately 1000 Å and the MW is 150 000. Because the persistence length of poly(γ-benzylL-glutamate) is 875 ( 100 Å55 and is on the same order as this helix length, it can be assumed that the helices in the film are straight and not bent. The helix orientation of γ-methyl-L-glutamate is 35° from the surface,14 which corresponds to a MW of 18 000 for a 110 Å film. The angle that the parallel β-sheet makes with the surface has not been determined; however, if we assume that the β-strand is oriented perpendicular to the substrate, the minimum MW of O-benzyl-L-tyrosine can be estimated. The axial distance between adjacent residues in the β-strand is 3.5 Å, so the minimum MW of an 800 Å O-benzyl-L-tyrosine film is 58 000. The MW of polymers in the antiparallel β-sheet and random coil conformations cannot be calculated because the strands may fold upon themselves, making it difficult to relate film thickness to MW. Spacing of Adjacent Polymers. R-Helices, β-sheets, and random coils have been observed in the grafted poly(amino acid) chains. The R-helix is composed of regularly spaced intramolecular hydrogen bonds between the backbone CdO of the n residue and the N-H of the n + 4 residue. The diameter of the helix can range between 9 and 26 Å,11,13 but the spacing of the initiators on the substrate does not have to correspond to this diameter for the formation of grafted R-helices.12,13,16 For β-sheets, intermolecular hydrogen bonds are formed between adjacent strands within antiparallel and parallel sheets. The initiation of the polymerization by the amino end groups on the surface orients all the chains in the same direction. The antiparallel β-structure can be formed when the chain end folds upon itself and forms hydrogen bonds with the earlier portion of the same chain segment, as shown in Table 3. However, the formation of parallel β-sheets requires that the spacing of adjacent chains be in the same range as the distance of the hydrogen bond. For a complete coverage of APS on a silicon substrate, the surface density56 of amino groups is 5 × 1014 cm-2. This corresponds to a molecular spacing of 4.47 Å per amino group or per polymer chain. The spacing between adjacent β-strands17,39 is between 4.7 and 4.9 Å. Since these values are similar to that for the spacing of the chains, the adjacent poly(amino acid) chains can hydrogen bond and form parallel β-sheets, as seen for poly(O-benzyl-Ltyrosine). Conclusion The vapor deposition polymerization method is an effective technique for producing a wide range of surfacegrafted polypeptides. For the deposition to occur, the NCA monomers have to be thermally stable, and aromatic pendant groups seem to give that stability. FTIR was used to determine the conformation of the polypeptides on the surface, and the resulting conformations matched those (55) Moha, P.; Weill, G.; Benoit, H. 1964, 61, 1240-1244. (56) Haller, I. J. Am. Chem. Soc. 1978, 100, 8050-8055.

Vapor Polymerization of Various R-Amino Acids

seen in bulk or in solution. The effect of reaction time on film thickness and conformation was also studied. The initial growth rate of vapor-deposited poly(γ-benzyl-Lglutamate) was approximately 100 times faster than for solution polymerization, and the poly(amino acids) with helical and parallel β-sheet conformations exhibited higher growth rates compared to those in the antiparallel β-sheet conformations. For poly(β-benzyl-L-aspartate) and poly(L-phenylalanine), a transformation from a random to a helical conformation was observed as film thickness increased. In addition to the polypeptides explored in this work, new poly(amino acids) can be synthesized by subsequent modification of the side chain to the already synthesized polymers on the surface. One reaction takes advantage of the ester functional group in poly(γ-benzyl-

Langmuir, Vol. 19, No. 4, 2003 1303 L-glutamate). By performing ester exchange reactions, polyglutamates with triethylene glycol, acid, and amino side groups have already been synthesized in our group.4,5 Through this study, we have shown that vapor deposition polymerization is a versatile method for the surface synthesis of poly(amino acids).

Acknowledgment. Financial support was provided by the NSF-MRSEC Program through the Center on Polymer Interfaces and Macromolecular Assemblies (Grant NSF-DMR 9808677) and the Stanford Graduate Fellowship program. LA020432I