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Grafting of Homo- and Block Co-polypeptides on Solid Substrates by an Improved Surface-Initiated Vapor Deposition Polymerization Yuli Wang and Ying-Chih Chang* Department of Chemical Engineering and Materials Science, University of California, Irvine, California 92697-2575 Received August 1, 2002. In Final Form: October 4, 2002 Grafting of poly(γ-benzyl L-glutamate) (PBLG) on silicon native oxide surfaces by the surface-initiated vapor deposition polymerization (SI-VDP) method was first demonstrated by Chang and Frank (Langmuir 1998, 14, 326). In the current study, we have further improved this method by redesigning the reaction chamber and optimizing the reaction parameters, including monomer concentration, substrate temperature, and reaction time. Through the process optimization, we can fabricate end-grafted R-helical PBLG films with tunable thicknesses from a few nanometers to hundreds of nanometers in less than 1 h of reaction time. For example, a 187 nm PBLG grafted film was synthesized at 95 °C monomer evaporating temperature, 75 °C substrate temperature, and 0.1 Pa in 30 min. In this study, the SI-VDP process was also applied to synthesize other homo-polypeptide and block co-polypeptide thin film systems. The successful syntheses of poly(γ-methyl L-glutamate) (PMLG), poly-L-phenylalanine (PLPA), poly(β-benzyl L-aspartate) (PBLA), poly(N-carbobenzyloxy L-lysine) (PCBL), PLPA-b-PBLG, and PBLG-b-PCBL grafted films were demonstrated. Their chemical compositions, secondary structures, thicknesses, refractive indices, and water contact angles were characterized by Fourier transform infrared spectroscopy (FTIR), ellipsometry, and contact angle goniometry, respectively.
1. Introduction Surface-grafted polypeptides have drawn considerable attention in recent years because of their distinct ordered secondary structures (such as R-helix and β-sheet). The ordered structures offer unique thin film properties. For example, the grafted poly(γ-benzyl L-glutamate) (PBLG) thin films on solid substrates demonstrated high electrooptical and piezoelectric efficiency, as a result of the presence of net dipole moment in the films.1,2 The grafted poly(L-glutamic acid) (PLGA) on membranes was employed as a pH-sensitive gating for water permeability because its conformational transition between helix and coil can be modulated by pH.3,4 Despite the interesting features that surface-grafted polypeptides may offer, research progress has been relatively slow compared to that for other polymeric materials. This is partially due to the lack of efficient synthetic methods to prepare polypeptide thin films with sufficiently high thickness and versatile chemical composition. Previous results from several research groups have concluded that the “grafting from” method, the surface-initiated ring-opening polymerization of N-carboxyanhydrides (NCAs) of amino acids, is by far the most effective approach for synthesizing high-density polypeptide films.5-11 In this approach, the polypeptide growth is (1) Jaworek, T.; Neher, D.; Wegner, G.; Wieringa, R. H.; Schouten, A. J. Science 1998, 279, 57. (2) Chang, Y.-C.; Frank, C. W.; Forstmann, G. G.; Johannsmann, D. J. Chem. Phys. 1999, 111, 6136. (3) Ito, Y.; Ochiai, Y.; Park, Y. S.; Imanishi, Y. J. Am. Chem. Soc. 1997, 119, 1619. (4) Ito, Y.; Park, Y. S.; Imanishi, Y. Langmuir 2000, 16, 5376. (5) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73. (6) Whitesell, J. K.; Chang, H. K.; Whitesell, C. S. Angew. Chem., Int. Ed. Engl. 1994, 33, 871. (7) Oosterling, M. L. C. M.; Willems, E.; Schouten, A. J. Polymer 1995, 36, 4463. (8) Heise, A.; Menzel, H.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M. Langmuir 1997, 13, 723. (9) Wieringa, R. H.; Schouten, A. J. Macromolecules 1996, 29, 3032.
initiated by a predeposited primary amine layer. The carboxyl anhydride groups of the monomer first react with the surface-bound amine groups to form amide bonds by releasing carbon dioxide molecules, leaving the free amines on the other end of the growing chains, which are then ready for the subsequent ring-opening reaction. However, several side reactions, such as the self-cyclization of the chains or the physical or chemical deactivation of the end groups, could terminate the polymerization prematurely. As a result, it was thought that the average degree of polymerization (DP) of PBLG synthesized through the NCA polymerization in solution phase was hardly over 300.12 Alternatively, Chang and Frank proposed to conduct the surface polymerization in gas phase.13 While following the same reaction mechanism as described above, the vaporized NCAs, instead of solvated NCAs, participated in the polymerization. The vapor phase reaction effectively suppressed the side reactions, hence greatly enhancing the reaction efficiency. For instance, a 42 nm PBLG film was synthesized under 0.01 Torr and 95 °C, in less than 4 h. It was postulated that the great reduction of impurities, the increase of the mean-free-path of the NCA evaporants, and the higher thermal energy of the NCA monomers were the major factors leading to the success of the vapor phase approach over the conventional solution phase reaction. In this study, we attempt to synthesize surface-grafted polypeptide films with controllable thickness, density, and composition. Specifically, we propose a general methodology that is applicable to a wide variety of polypeptide (10) Chang, Y.-C.; Frank, C. W. Langmuir 1996, 12, 5824. (11) 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. (12) Kricheldorf, H. R. R-Aminoacid-N-carboxyanhydrides and related heterocycles; Springer-Verlag: Berlin, Germany, 1987. (13) Chang, Y.-C.; Frank, C. W. Langmuir 1998, 14, 326.
10.1021/la026343n CCC: $22.00 © 2002 American Chemical Society Published on Web 11/08/2002
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Scheme 1. Schematic Diagram of the Experimental Setup for the Vapor Deposition Polymerization
materials. The previous proof-of-concept vapor phase process serves as the foundation of the current work. To distinguish from the conventional “vapor deposition polymerization” process, which is often referred to as a nongrafting film deposition process, we call our current process “ surface-initiated vapor deposition polymerization” (SI-VDP) to emphasize the grafting nature of the resulting films. Previously, experiments have shown that the temperature, vacuum, reaction time, and monomer concentration are the key factors influencing the grafting efficiency of the SI-VDP process.13 In this study, these parameters will be further evaluated in a redesigned reaction chamber. In addition, new types of homopolypeptide thin films, including poly-L-phenylalanine (PLPA), poly(β-benzyl L-aspartate) (PBLA), and poly(N-carbobenzyloxy L-lysine) (PCBL), as well as the diblock copolypeptide thin films, including PLPA-b-PBLG and PBLG-b-PCBL, will be fabricated via the improved protocol. To verify the synthetic results, both ellipsometry and Fourier transform infrared spectroscopy (FTIR) will be used to characterize the film thicknesses, refractive indices, chemical compositions, and secondary structures. 2. Experimental Section 2.1. Materials. The NCAs of γ-benzyl L-glutamate (BLG), γ-methyl L-glutamate (MLG), L-phenylalanine (LPA), β-benzyl L-aspartate (BLA), and N-carbobenzyloxy L-lysine (CBL) were synthesized by phosgenating the corresponding amino acids with triphosgene. They were then purified by rephosgenation according to the procedure published by Dorman et al.14 All of the synthetic operations were carried out in a dry glovebox. (3-Aminopropyl)triethoxysilane (APS) was used as the initiator. All chemicals were purchased from Sigma-Aldrich Chemicals (Milwaukee, WI) and used as received. Undoped, double-polished, wedged silicon (100) wafers (Harrick Scientific Corp., with a wedge angle of 0.25° and dimensions of 10/16 in. × 7/16 in. × 500 µm) were used as the substrates. On average, the thickness of the silicon native oxide layer measured with an ellipsometer was 11.4 ( 0.2 Å. 2.2. Cleaning of Substrates. The substrates were cleaned with a freshly prepared mixture of concentrated sulfuric acid and 30% hydrogen peroxide (70/30, v/v) at 120 °C for 30 min. They were then washed with a large amount of distilled water and subsequently rinsed with acetone. The substrates were blown dried under a stream of nitrogen and immediately used in the silanization step. 2.3. Silanization of Substrates. The formation of the APS layer on the substrates was carried out in the vapor phase under reduced pressure. The procedures were adapted from Chaudhury (14) Dorman, L. C.; Shiang, W. R.; Meyers, P. A. Synth. Commun. 1992, 22, 3257.
and Whitesides’ with slight modification.15 The cleaned substrates and a small vial (Fisher brand 12 mm × 35 mm, 1/2 DR) containing 150 µL of APS were placed on a Petri dish inside a vacuum desiccator (Wheaton dry-seal desiccator, 100 mm). The desiccator was evacuated down to about 40 Pa with a vacuum pump. At this point, the desiccator was disconnected from the vacuum pump. It was tightly sealed and kept in this condition for about 16 h at room temperature. After the completion of silanization, the substrates were taken out from the desiccator. They were ultrasonicated in fresh acetone for 5 min, repeated for 5 times, and finally dried under a stream of nitrogen. The thickness of the immobilized APS layer measured with an ellipsometer was 17.6 ( 0.7 Å. 2.4. Surface-Inititiated Vapor Deposition Polymerization. 2.4.1. Chamber Configuration. The reaction chamber used in the SI-VDP procedure was redesigned from the previous version of Chang and Frank’s. The schematic diagram of the SI-VDP setup is shown in Scheme 1. The cylindrical vacuum chamber (internal dimensions: diameter × height ) 175 mm × 160 mm) was made of aluminum. The monomer (typically 8 mg of NCA) was first dissolved in 0.3 mL of anhydrous tetrahydrofuran and was poured into an aluminum sample container (internal dimensions: length × width × height ) 33 mm × 13 mm × 4 mm). The solution was quickly dried in a vacuum so that a thin and uniform layer of NCA was spread on the bottom of the container. Subsequently, a substrate was placed atop of the sample container with the APS-modified side facing downward, thus forming an internal cell (as shown in Scheme 1). The displacement between the monomer source and the substrate was about 4 mm. This cell was then sandwiched by two heating plates: one beneath the monomer source and the other above the substrate with immediate contacts. The bottom heating plate provided the heat source to evaporate the NCA species, and the top one controlled the substrate temperature with typical temperatures from 20 to 110 °C. Although the internal cell is not tightly sealed, this setup promotes the chance that NCA vapor would first encounter the initiator-modified substrate for polymerization, thus increasing the effective concentration locally. In other words, with this setup, we hope to reduce the amount of NCA vapor to be evacuated to the outer chamber. 2.4.2. Reaction Conditions of SI-VDP. To start the reaction, the chamber was evacuated down to 0.1-0.3 Pa, and then the temperature of the top heating plate was raised to a preset value and stabilized for 3 min. Consequently, the temperature of the bottom heating plate was raised to the melting point temperature of the corresponding NCA. When the bottom temperature reached the preset value, the reaction was timed. During the reaction, the chamber was continuously evacuated to maintain a constant vacuum. However, the internal cell has a higher vapor pressure built up due to the NCA evaporation. The reaction proceeded for a controlled time period, typically from 3 to 60 min. Note: Throughout the text, the two temperature settings will be (15) Chaudhury, M. K.; Whitesides, G. M. Langmuir 1991, 7, 1013.
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Scheme 2. Schematic Representation of the Synthesis of Surface-Grafted Polypeptides and Diblock Copolypeptidesa
a Key: (a) Silanization of the substrate with APS to form an initiator layer; (b) SI-VDP of vaporized NCA monomer 1 to form a homopolypeptide thin film; (c) SI-VDP of vaporized NCA monomer 2 to form a diblock copolypeptide thin film. The chemical formulas of side groups R1 and R2, and the corresponding polypeptide, are listed in Table 1.
expressed as, for example, 60 °C/95 °C, to indicate the top plate temperature (substrate temperature) at 60 °C and the bottom plate temperature (monomer evaporating temperature) at 95 °C, respectively. 2.4.3. Cleaning Treatment of the Surface Films. After the completion of the reaction, all samples were removed from the chamber and cleaned with a mixture of dichloroacetic acid (DCA) and chloroform (20/80 (v/v)) in an ultrasonic bath for at least 5 min, before being rinsed with fresh chloroform and dried under a stream of nitrogen. The DCA/chloroform mixture can dissolve polypeptide materials with various Mw values effectively and, hence, can be routinely used as a cleaning reagent to discern loosely bound physisorbed polypeptides from the surface-bound polypeptides, as demonstrated in previous publications.8,9,11,13 In our case, an additional 5-min ultrasonication was applied simultaneously to promote the cleaning efficiency, and another good solvent, chloroform, was also applied alternatively to further clean the surfaces. To prove the effectiveness of the cleaning treatment, additional cleaning, by strong acids such as pure DCA or trifluoroacetic acid that disrupts H-bonding in polypeptide chains, and 24 h of Soxhelet extraction in chloroform were also conducted for comparison. No appreciable difference was found from these approaches. 2.4.4. Block Copolymerization by Sequential SI-VDP. To create a block copolypeptide thin film, the previously synthesized polypeptide thin film (after it was cleaned with a DCA/chloroform mixture) was first immersed in a pH 11 buffer solution (10 mM sodium phosphate) for 1 min to convert the N-terminal groups from -NH3+ to -NH2. It was then washed with acetone and chloroform and used as the substrate for the second grafting polymerization following the same procedure outlined above. 2.5. Optical Ellipsometry. Ellipsometric measurements were carried out with a Gaertner LSE stokes ellipsometer using a He-Ne laser of wavelength of 632.8 nm and an incident angle of 70°. The Gaertner Ellipsometer Measurement Program was used to calculate the film thickness and refractive index. At least 10 measurements were made to average the thickness and/or refractive index on each surface. The optical constants of silicon were fixed with Ns ) 3.882 and Ks ) -0.0019. An isotropic twophase model (Si/film) was used for calculation. If the film (SiOx + organic film) thickness was less than 5 nm, a fixed film refractive index Nf ) 1.462 was used to calculate the film thickness. If the film thickness was larger than 5 nm, thickness and refractive index can be calculated independently. The experimental thickness of the grafted polypeptide films was obtained by subtracting the thicknesses of SiOx (11.4 Å) and APS (17.6 Å) from the calculated thickness.
2.6. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra of the grafted polypeptide films on the silicon wafers were recorded on a Nicolet Magna-IR 860 spectrometer in transmission mode with a clean silicon wafer as the reference. Before and during the measurements, the sample chamber was purged with dry air (Whatman FT-IR purge gas generator). Spectra were recorded at 4 cm-1 resolution, and 32 scans were collected. To yield quantitative data, deconvolution and iterative curvefitting were carried out in the ester and amide I region (16001750 cm-1) with the Peaksolve software (version 1.05, Galactic Industries Corp.). The deconvolution was applied with a mixed ratio of Lorentzian and Gaussian line-shape distributions. The frequencies of the band centers were used as initial input parameters for the curve-fitting procedure, and all intensities were iterated to obtain a minimum rms error. The fraction of secondary structures (R-helix and β-sheet) was evaluated by the relative areas of the deconvoluted peaks.16
3. Results and Discussion The strategy to synthesize grafted polypeptide films through ring-opening polymerization of NCAs is summarized in Scheme 2. The bare silicon wafer was first silanized by APS to create an initiator layer (Scheme 2a). The vaporized NCA monomer 1 in contact with the surface initiator was thereby initiated to form a monolayer of polypeptide film with a DP of m (Scheme 2b). To create a diblock copolypeptide film, the homopolypeptide film was cleaned and then used as the macroinitiator to polymerize the NCA monomer 2 (Scheme 2c). In principle, this procedure can be repeated to produce multiple blocks of copolypeptide films. In this study, the homo- and block co-polypeptide species selected for the proof-of-concept purpose are summarized in Table 1. 3.1. Optimization of the SI-VDP Process. In the previous study, Chang and Frank developed the SI-VDP method to graft R-helical PBLG films onto solid substrates.13 They found that the termination reactions that normally occurred in solution phase were greatly suppressed in vacuo. As a result, the thickness of the PBLG film prepared by the SI-VDP method significantly exceeded the thickness obtained by other grafting approaches. (16) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469.
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Table 1. Chemical Formulas of NCA Side Groups R1 and R2, and the Corresponding Polypeptide Materials Fabricated in This Study
In Chang’s study, the reaction chamber was heated in a preheated oven; hence, the temperature distribution inside the flask could not be directly controlled and measured. In the current work, we added two separate heating components and thermocouples immediately adjacent to the substrate and the monomer source to directly control and measure their respective temperatures. Using this redesigned reaction chamber, we further evaluated two reaction parameters which were neglected previously: the monomer concentration and the substrate temperature. PBLG was used as the model system in order to compare our results directly with the literature. 3.1.1. Monomer Concentration Effects. In the solution phase surface-initiated polymerization, it is known that the monomer concentration influences the film growth. For example, Wieringa et al. found that higher NCA concentration (in the range between 0 and 2 mol/L) led to thicker polyglutamate films,11 and the ideal monomer concentration was 0.1-0.5 mol/L. In the SI-VDP process, the monomer concentration is low if a relatively large vacuum chamber is used. For instance, 10 mg of BLGNCA can only produce 1.2 × 10-5 mol/L in concentration in a 3-L chamber. This concentration is 4 orders of magnitude less than that used in the solution phase polymerization. To compensate for the concentration loss, we introduced a small container inside the vacuum chamber to confine the NCA vapor within a smaller space, as described in section 2.4.1. The confined space (∼1.7 mL) localized the vapor of the NCA monomers, promoting the effective concentration locally in contact with the initiator-modified substrate. As a result, the NCA concentration is estimated to be around 0.02 mol/L when 10 mg of BLG-NCA is used. We conducted SI-VDP of BLG-NCA at various concentration levels. When a small amount of BLG-NCA was used (for example, 1 mg (0.002 mol/L) of BLG-NCA), a thin and inhomogeneous PBLG film resulted. On the other hand, the use of an excess amount of BLG-NCA (for example, 20 mg (0.04 mol/L) of BLG-NCA) led to a higher evaporation rate than the polymerization rate; as a result, a thick, unreacted BLG-NCA layer that condensed on the substrate prevented the surface reactive
groups from further polymerization with vapor NCA. We found that the optimal amount of BLG-NCA monomer for our experimental setup was between 7 and 9 mg (i.e. 0.015-0.019 mol/L). Therefore, unless otherwise noted, 8 mg of BLG-NCA monomer source is the amount used in all experiments. 3.1.2. Substrate Temperature Effects. Another new feature of the modified SI-VDP process is the inclusion of a second heating plate behind the substrate to control the substrate temperature. Figure 1 shows the transmission FTIR spectra of PBLG films obtained by SI-VDP before and after the cleaning procedure, when the substrate temperatures are at 25 °C (1a) and 95 °C (1b), respectively. It is known that the three peaks at 1860, 1786 (the anhydride carbonyl), and 1733 cm-1 (the benzyl ester) are the typical peaks of the BLG-NCA monomer, while the peaks at 1733 (the benzyl ester from the PBLG side groups, which overlaps with the benzyl ester peak of NCA), 1653 (the amide I of R-helical PBLG), and 1547 cm-1 (the amide II of R-helical PBLG) are the characteristic peaks of the PBLG. By identifying the peak locations on the spectra, the conversion from the NCA monomers to the grafted polypeptide chains can be verified. When the substrate temperature is 25 °C, the FTIR spectrum in Figure 1a-(1) suggests that the film before the cleaning treatment contains both monomer reactants and polypeptide products. Following the cleaning treatment described in section 2.4.3, the unreacted NCA monomers were effectively removed, since no characteristic peaks of BLG-NCA are present in the spectrum shown in Figure 1a-(2). We can estimate the effective molar fraction of surface-deposited monomers that convert to the grafted PBLG chains by taking the ratio of the benzyl ester peak at 1733 cm-1 after and before the cleaning treatment. By the peak deconvolution, the conversion fraction from the surface-deposited NCA to the grafted PBLG is about 24%. Interestingly, when the substrate temperature is raised to 95 °C, as estimated from Figure 1b, the conversion fraction is 51%, which is two times higher than that of the sample with the substrate temperature at 25 °C. This strongly suggests that more NCA monomers are converted
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Figure 2. Dependence of PBLG film thickness on the substrate temperature.
Figure 1. Transmission FTIR spectra of PBLG films obtained by SI-VDP before and after the cleaning procedure. The substrate temperatures are (a) 25 °C and (b) 95 °C. The monomer evaporating temperature is 95 °C, and the reaction time is 30 min.
to the grafted PBLG chains when the substrate temperature is elevated. This observation is further confirmed by the ellipsometric measurements, where the thickness of 102.5 ( 0.8 nm at 95 °C is significantly higher than that of 77.0 ( 1.6 nm at the 25 °C substrate temperature. As a control experiment, we repeated SI-VDP on an octadecyltrichlorosilane (OTS) modified silicon substrate at 95 °C/95 °C, for a 30 min reaction time. After cleaning, there is no polypeptide found on the surface by both FTIR and ellipsometer. This supports the notion that it is the surface amine sites that initiate PBLG growth and confirms the effectiveness of the cleaning treatment in removing unbounded materials. (a) Optimization of the Substrate Temperature. To further verify the optimal substrate temperature to create surface-grafted PBLG films, we conducted SI-VDP at different substrate temperatures when other parameters were fixed. Figure 2 shows the thicknesses of the grafted PBLG films prepared at different substrate temperatures from 25 to 105 °C. The optimal substrate temperature was found to be between 50 and 75 °C, at which it produced the thickest PBLG monolayer film by far. For instance, a PBLG film with a thickness of 187.4 nm was synthesized when the substrate temperature was 75 °C.
The FTIR spectra indicate that all of the PBLG films adopt R-helical conformations (as identified by the peak locations of the amide I and II bands). A number of publications have shown that the surface-bound R-helical PBLG (synthesized by either “grafting to” or “grafting from”) tends to tilt toward the substrate surface, with the average tilt angles 〈θ〉 33-65° from the surface normal.17-19 For a rough estimation, because each repeating unit contributes 0.15 nm along the helical molecular axis,20 the average DP of a 187 nm thick PBLG film is about 1700 (i.e. Mw ∼ 3.7 × 105). The extended lifetime of the vapor and less condensation on the surfaces may contribute to the high conversion from NCA to PBLG when the substrate temperature is elevated above room temperature. However, the continuous increase of the substrate temperature is not favorable. In one extreme case, when the substrate temperature is elevated to 140 °C, no film growth occurs. This might be explained by the following postulates: (1) The physical hindrance caused by the randomization of molecular chains at high substrate temperature. We found that when the substrate temperature was higher than 80 °C, the orientation of the grafted PBLG chains became randomized. The higher the temperature was, the faster the randomization process became. The randomization can be completed within 1 min above 130 °C.21 Since the randomization process reduces the persistent length of the molecular chain and produces less ordered surface structures, the amine end groups become less accessible for the further ring-opening reaction, therefore resulting in low DP. (2) The reduction of the effective NCA concentration at surfaces. When the substrate temperature is higher than that of the vaporized NCA, the NCA molecules bombarded on the substrate may have a shorter retention time, and thus, the effective NCA concentration is decreased. (17) Higashi, N.; Koga, T.; Niwa, M. Langmuir 2000, 16, 3482. (18) Wieringa, R. H.; Siesling, E. A.; Werkman, P. J.; Angerman, H. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17, 6485. (19) Williams, A. J.; Gupta, V. K. J. Phys. Chem. B 2001, 105, 5223. (20) Samulski, E. T. Liquid Crystalline Order in Polypeptides; Samulski, E. T., Ed.; Academic: New York, 1978; pp 167-190. (21) By using a solvent treatment method, we can get unidirectionally aligned PBLG films with tilt angles 〈θ〉 as small as 5°. The thermally induced randomization of this oriented PBLG film was studied. Above 80 °C the PBLG film began to be randomized. When the film was treated at 130 °C for 1 h, the 〈θ〉 was increased to 48°. The manuscript is currently in preparation.
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Table 2. Reaction Conditions, Ellipsometric Results, and Contact Angle Data for Surface-Grafted Polypeptidesa polypeptide
NCA amount (mg)
temp setting (°C/°C)
reaction time (min)
film thickness (nm)
refractive index
water contact angleb (deg)
PLPA PMLG PBLA PCBL
8 8 8 8
98/98 98/98 100/135 102/102
30 30 30 30
43.6 ( 0.9 69.8 ( 1.0 79.0 ( 1.5 99.3 ( 0.3
1.534 ( 0.005 1.486 ( 0.005 1.506 ( 0.005 1.557 ( 0.001
76 ( 2 55 ( 2 67 ( 2 62 ( 2
a The transmission FTIR spectra of the corresponding polypeptides are shown in Figure 4. b Water static contact angle. The angle at both sides of the free-standing droplet was measured at five different spots of the same substrate. Given results are average values of all measurements.
Figure 3. Progression of the grafted PBLG film thickness monitored by ellipsometry. The reaction conditions are shown in the inserted box.
(b) Homogeneity of the Films. The standard deviation of the thicknesses of eight samples shown in Figure 2 is less than 5 nm in a 1 cm × 1 cm area for at least 80 different measurements, indicating that the obtained PBLG film is very homogeneous in the whole area. 3.1.3. Reaction Rate. Figure 3 shows the PBLG growth as a function of time at one of the most optimal conditions (8 mg BLG-NCA, 60 °C/95 °C). In this particular case, the PBLG growth reaches a plateau with the film thickness of 160 nm in 20 min. Compared to the previous optimal results from Chang and Frank where the film reached the plateau at the thickness of 42 nm for 4 h of reaction time,13 our current results show a significant improvement in both reaction rate and the resulting film thickness. The slow growth of the film after 30 min might be due to the blockage of surface initiator sites that prevent the further polymerization. Nevertheless, using Figure 3 as the calibration standard, we can obtain any desirable film thickness between 3 and 160 nm by adjusting the reaction time. 3.2. Synthesis of Other Grafted Polypeptide Thin Films by SI-VDP. To further extend the applicability of the modified SI-VDP method, the fabrication of various types of polypeptide thin films was examined. As summarized in Table 1, poly-L-phenylalanine (PLPA), poly(γ-methyl L-glutamate) (PMLG), poly(β-benzyl L-aspartate) (PBLA), and poly(N-carbobenzyloxy L-lysine) (PCBL) grafted thin films were synthesized. The corresponding reaction conditions and the resulting film properties, including the thicknesses, refractive indices, and water contact angles, are listed in Table 2. The FTIR spectra of the corresponding films are shown in Figure 4. From the FTIR spectra in Figure 4, the chemical compositions of the surface-grafted polypeptide films can be identified by their characteristic peaks and are
Figure 4. Transmission FTIR spectra of surface-grafted polypeptides: (a) PLPA; (b) PMLG; (c) PBLA; (d) PCBL. The reaction conditions, film thicknesses, refractive indices, and water contact angles are listed in Table 3. Table 3. FTIR Spectral Peak Assignment for Surface-Grafted PLPA, PMLG, PBLA, and PCBL amide I (cm-1) amide A Rβpolypeptide (cm-1) helix sheet PLPA PMLG PBLA PCBL
3287 3294 3302 3297
1661 1654 1666 1652
1642 1628 1639
amide II (cm-1) side Rβgroup helix sheet (cm-1) 1539 1549 1556 1544
1495 1517 1537
1738 1736 1696
consistent with the literature. The secondary conformations of the films are identified by the location of the amide I and the amide II bands, as summarized in Table 3. We found that the surface-grafted PCBL (99 nm) adopts a pure R-helix conformation; surface-grafted PLPA (44 nm), PMLG (70 nm), and PBLA (79 nm) films adopt R-helix predominantly, as well as a small portion of β-sheet. The molar fraction of β-sheet can be estimated from the relative areas of the deconvoluted peaks in the amide I region. The β-sheet fraction is 13% for PMLG, 18% for PBLA, and 17% for PLPA, respectively. It is known that many polypeptide materials precipitate in the reaction media that are used to dissolve their NCA monomers. For example, PLPA, a typical hydrophobic polypeptide, precipitates in tetrahydrofuran and N,Ndimethylformamide. As a result, the grafting efficiency and the resulting molecular weight are low. On the contrary, the SI-VDP process in principle can circumvent the solubility problem, since no solvent is required at all. Although we did not fully optimized the reaction parameters for each polypeptide system, the results have suggested the feasibility and high efficiency of the SIVDP protocol for all the systems we examined, allowing us to further investigate the individual film properties.
Grafting of Homo- and Block Co-polypeptides
PMLG is an interesting polypeptide in that its conformation is subject to change, adopting either R-helical or β-sheet forms, depending upon its environment history and molecular weights.22-24 For the PBLA film, the amide I absorption is around 1666 cm-1, indicating an R-helical conformation. It is an interesting polypeptide material because its helical structure can be switched between a right- and left-handed helix by the solvent and thermal treatments,25,26 whereas other polypeptide materials in this study always adopt a right-handed helical structure optically. The removal of the carbobenzyloxy side groups converts PCBL to poly(L-lysine) (PLL), a hydrophilic polypeptide having important functions. Therefore, the successful synthesis of surface-grafted PCBL thin films leads to many exciting applications that involve PLL-modified surfaces. Similarly, we anticipate that the chemical derivations of side groups of the surface-grafted polypeptides, such as transesterification and amidation, allow conversion from one polypeptide to another. We will report the results of the postmodification of polypeptide films in the future. 3.3. Synthesis of Block Copolypeptides by Sequential SI-VDP. Recently, the first case of the synthesis of a grafted diblock copolypeptide, PBLG-b-PMLG, in solution phase was reported by Wieringa et al.27 By stopping the first polymerization and washing away the nongrafted materials, a second block of PMLG was initiated from the N-termini of the PBLG chains through the renewed polymerization. Although the combined film thickness was less than 30 nm, the feasibility was confirmed by FTIR, ellipsometry, and X-ray photoelectron spectroscopy (XPS). As pointed out in the previous text, the side reactions in the SI-VDP process are suppressed, so the SI-VDP is anticipated to be a better protocol to synthesize block copolypeptides than the conventional solution phase reaction. The reaction protocol and the detailed procedures to synthesize block copolypeptides have been described in Scheme 2 and section 2.4, respectively. 3.3.1. Renewed Polypeptide Growth. At first we test the ability of renewed polypeptide growth by repeating the SI-VDP process of BLG-NCA. Figure 5 shows the transmission FTIR spectra of the PBLG films formed during three consecutive SI-VDP processes. The increases of both IR absorbance and the cumulative ellipsometric thickness as labeled above each spectrum are the evidences of the addition of the PBLG material from each cycle. The thorough washing procedure after each cycle excludes the possibility of physisorption of the PBLG species; hence, we conclude that the successful growth of the grafted PBLG film is indeed through the renewed SI-VDP process. The renewed PBLG growth indicates that the initiating sites on the top of the growing chains remain active after the cleaning procedure. (This also testifies that the side reactions, such as the termination reaction caused by the nucleophilic addition of terminal amine to the 2-CO group of NCA, are suppressed.) The cleaning procedure removes unreacted NCAs and helps the initiating sites be exposed. However, the exposure of the initiating sites is not perfect because the PBLG chains are tilted on the surface, (22) Blout, E. R.; Asadourian, A. J. Am. Chem. Soc. 1956, 78, 955. (23) Baier, R. E.; Zisman, W. A. Macromolecules 1970, 3, 70. (24) Kugo, K.; Okuno, M.; Kitayama, K.; Kitaura, T.; Nishino, J.; Ikuta, N.; Nishio, E.; Iwatsuki, M. Biopolymers 1992, 32, 197. (25) Karlson, R. H.; Norland, K. S.; Fasman, G. D.; Blout, E. R. J. Am. Chem. Soc. 1960, 82, 2268. (26) Bradbury, E. M.; Downie, A. R.; Elliott, A.; Hanby, W. E. Proc. R. Soc. (London) 1960, A259, 110. (27) Wieringa, R. H.; Siesling, E. A.; Werkman, P. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17, 6491.
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Figure 5. Transmission FTIR spectra of the grafted PBLG thin film after (a) the first, (b) the second, and (c) the third cycle of SI-VDP of BLG-NCA. The film thickness determined with an ellipsometer is indicated in each cycle. Reaction conditions: BLG-NCA 8 mg, 60 °C/95 °C, 6-min reaction time for the first cycle, 10-min for the second cycle, and 30-min for the third cycle.
Figure 6. Transmission FTIR spectra of (a) a PLPA film (38 nm) after the first cycle of SI-VDP of LPA-NCA (reaction conditions: LPA-NCA 8 mg, 98 °C/98 °C, 20 min) and (b) a diblock copolypeptide PLPA-b-PBLG film (59 nm) after the second cycle of SI-VDP of BLG-NCA (reaction conditions: BLG-NCA 8 mg, 60 °C/95 °C, 30 min). (c) FTIR spectrum obtained by subtracting part a from part b, corresponding to the spectrum of the second PBLG block (21 nm).
reducing the grafting efficiency in the next SI-VDP process. Therefore, longer time is needed for the second SI-VDP to achieve the same thickness film growth. For example, we can graft 60 nm of PBLG film in the first SI-VDP cycle (6-min reaction time) and 44 nm in the second cycle (10min reaction time) and 39 nm in the third cycle (30-min reaction). 3.3.2. Synthesis of Diblock PLPA-b-PBLG. The sequential addition of two different NCA monomers results in the formation of a diblock copolypeptide film. As an example, a diblock PLPA-b-PBLG copolypeptide film was synthesized by the sequential SI-VDP of LPA-NCA and BLG-NCA. Figure 6 shows the transmission FTIR spectra of the 38-nm PLPA film after the first cycle of SI-VDP of LPA-NCA and the 59-nm PLPA-b-PBLG diblock film after the second cycle of SI-VDP of BLG-NCA. In addition,
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density of each layer, however, have not been identified. It is quite likely that some of the polypeptide molecules in the first block might not participate in the renewed polymerization because of the mismatched diameters of different helical rods or the burial of the amine end groups of the chains; therefore, it might result in a lower-density second layer. Design of the grafting density of the first layer spaced at distances consistent with the sterical requirement of the second layer may be required to improve the yields.5
Figure 7. Transmission FTIR spectra of (a) a PBLG film (60 nm) after the first cycle of SI-VDP of BLG-NCA (reaction conditions: BLG-NCA 8 mg, 60 °C/95 °C, 6 min) and (b) a diblock copolypeptide PBLG-b-PCBL film (115 nm) after the second cycle of SI-VDP of CBL-NCA (reaction conditions: CBL-NCA 8 mg, 102 °C/102 °C, 60 min).
the successful addition of PBLG from the PLPA layer is also supported by the FTIR spectra, where the appearance of the benzyl ester peak at 1733 cm-1 can only be attributed to the PBLG block, and the amide I peak shifts from 1661 to 1655 cm-1. The subtraction of the PLPA spectrum from the PLPA-b-PBLG spectrum results in the spectrum of the R-helical PBLG (having the amide I peak at 1653 cm-1). This clearly illustrates the formation of a diblock PLPAb-PBLG. 3.3.3. Synthesis of Diblock PBLG-b-PCBL. We further demonstrate the synthesis of a diblock PBLG-b-PCBL copolypeptide film by the sequential SI-VDP of BLGNCA and CBL-NCA. Figure 7 shows the transmission FTIR spectra of the PBLG block (60 nm) after the first SI-VDP of BLG-NCA and the diblock copolypeptide PBLG-b-PCBL (115 nm) after the second SI-VDP of CBLNCA. In addition to the thickness growth monitored by ellipsometry, the appearance of a carbobenzyloxy peak at 1696 cm-1 (after deconvolution and curve-fitting procedures) proves the success of grafting of PCBL on top of PBLG. At present, the grafting efficiency is estimated on the basis of the increase of the overall thickness growth and the characteristic IR peaks of the films after each polymerization cycle. The polydispersity and the grafting
4. Conclusions The surface-initiated vapor deposition polymerization method was significantly improved by redesigning the vacuum chamber and optimizing the reaction parameters. We found that, in addition to the previously known factors, the monomer concentration and the substrate temperature also played significant roles in the grafting efficiency. Under one of the optimal conditions for the synthesis of PBLG films with the substrate temperature between 50 and 75 °C, the evaporating temperature at 95 °C, and the pressure under 0.1 Pa, the growth of the films can be tuned from 3 nm to near 200 nm within 30 min. The SI-VDP process can be extended to synthesize various polypeptides with ordered secondary structures, such as PLPA, PMLG, PBLA, and PCBL. The SI-VDP process provides a general methodology to prepare surfacegrafted polypeptides with by far the highest thickness and density. By using sequential SI-VDP processes, block copolypeptide thin films, such as PLPA-b-PBLG and PBLG-b-PCBL, can be synthesized. Thus, the SI-VDP process can be utilized to construct multilayered surface-grafted films with tuned chemical properties. To summarize, the current version of SI-VDP has proven its potential by further improving itself. By taking consideration of the substrate temperature and the monomer concentration, we can synthesize polypeptide thin films with thicknesses almost 1 order of magnitude higher than those from any existing methods, including its previous version. More importantly, it opens up opportunities for researchers to investigate a wide variety of thin films made of polypeptide materials, which only now has become available. We have pointed out several unique polypeptide films created by the SI-VDP process and will report their properties in the future. Acknowledgment. We thank Peter Z. Shi for manufacturing the aluminum vacuum chamber used for vapor deposition polymerization. LA026343N
