glutamates Grafted from Self-Assembled Monolayers - American

Chapter 10

Factors Influencing the Layer Thickness of Poly-L-glutamates Grafted from Self-Assembled Monolayers 1

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H. Menzel , A. Heise , Hyun Yim , M . D. Foster , R. H. Wieringa , and A. J. Schouten Downloaded via TUFTS UNIV on July 11, 2018 at 11:13:41 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Institut für Makromolekulare Chemie, Universität Hannover, Am Kleinen Felde 30, 30167 Hannover, Germany Institute of Polymer Science, University of Akron, Akron, Ohio 44325-3909 Department of Polymer Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands 2

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Factors influencing the thickness of polypeptide layers grafted from self-assembled monolayers were investigated by varying the initiator site density and the reactivity of the N-carboxyanhydride monomer. To vary the density of initiating sites and to match the steric requirements of the growing polymer chain, mixed self-assembled monolayers were prepared with terminal amino groups. These SAMs were prepared by coadsorption of bromine- and methyl-terminated silanes and a subsequent in-situ modification. Polymerization experiments with these SAMs reveal an influence of initiator site density on the polymer layer thickness. SAMs prepared with 60% functionalized silane showed an optimum thickness. The variation in layer thickness due to the variation of initiator site density is smaller than the influence of the monomer reactivity. α-helical polypeptides like poly-L-glutamates are rigid rod-like polymers and have several interesting properties. Most notably, they have a large dipole moment along the helix axis (7). The antiparallel orientation of the rods is the energetically favored arrangement and the dipole moments of the individual polymer rods are compensated. Approaches to an unidirectional orientation, which would result in a net dipole moment of the polymer film, are casting the polymer in a strong magnetic field (2) or tethering the polymer backbone to a surface and achieving a high density of polymer chains (3 9). The tethering of the polymers to a surface has been performed by grafting preformed polymers onto the surface (3 - 5, 70) as well as by grafting the polymer from the surface (6 - 8, 10). The latter method has the advantage that a higher grafting density can be achieved because only small monomelic molecules have to diffuse to the surface and there is no blocking of binding sites by already tethered material. On the

©1998 American Chemical Society

Frank; Organic Thin Films ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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132 other hand, the surface the polymer is grafted from has to have appropriate initiating sites, which have to be introduced first. Furthermore, the polymer grafted from a surface is hard to characterize, and there is no chance to fractionate the polymer or to separate oligomers with a different secondary structure as they may occur in the polypeptide synthesis (77). Therefore, special attention has to be paid to the polymerization conditions in order to yield homogeneous films.

Mechanism of the NCA-Polymerization In order to understand the requirements and limitations of a grafting from polymerization it is worthwhile to have a closer look at the mechanism. Poly-Lglutamates can be polymerized from the corresponding N-carboxyanhydride 1. If the polymerization is initiated by primary amines or secondary amines without any steric hindrance, the polymerization proceeds via the "amine mechanism" (Figure 1) (77 73).

5 Figure 1: Initiation and propagation in the NCA-polymerization via the amine-mechanism (According to ref. 12) The amine attacks the NCA-ring and ring opening occurs. The intermediate 3 eliminates carbon dioxide and a dimer with an amino endgroup is generated, which can attack the next monomer. The new amino group is less reactive than a primary amine. Therefore, the initiation is much faster than the propagation and the degree of polymerization can be adjusted to some extent by varying the monomer-to-initiator ratio (77 - 73). In this respect, the N C A polymerization has a kind of "living character". (In a living polymerization all chains are started simultaneously because the initiation is much faster than the propagation and there are no terminating side reactions. The latter condition is not fulfilled for the NCA-polymerization.) The amine polymers can be synthesized (14, 15) or tethered to a surface if the amino group is attached.


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A further mechanism which is discussed in literature, is the "carbamate mechanism" (Figure 2). The intermediate carbamic acid 3 is deprotonated by the initiating amine or the terminal amino group of the growing chain and further reaction takes place via the deprotonated carbamic acid 3 . The nucleophilic attack results in the formation of an intermediate anhydride 6, which decarboxylates. A new peptide bond is formed and the polymer chain has been prolonged by one monomelic unit. f

Figure 2: Initiation and propagation in the NCA-polymerization via the carbamate mechanism (According to ref. 12) Strong bases can deprotonate the N-carboxyanhydride 1. The deprotonated anionic monomer Γ is a strong nucleophilic agent which can attack a monomelic N C A and, therefore, is an "activated monomer". The attack of a N C A ring results in the intermediate 8 (Figure 3). The intermediate 8 has two reactive sites: i) the electrophilic N-acyl group and ii) the nucleophilic carbamate group. The latter one can react according to the mechanism shown in Figure 2 (pathway A in Figure 3). Furthermore, this group can decarboxylate and the generated amine end group can react according to the amine mechanism shown in Figure 1 (pathway Β in Figure 3). Since the decarboxylation intermediate 9* is an amide anion, the decarboxylation also results in deprotonation (activation) of a monomer molecule. This activated monomer can now attack a N C A ring either of a monomer or at the end of a growing chain (pathway C in Figure 3). The polymerization is much faster if strong bases or sterically hindered amines are used as initiators. Therefore, it was concluded that the attack of the activated monomer (pathway C in Figure 3) is much faster than the other possible polymerization reactions (77). The diverse propagation steps encountered with this mechanism have consequences. First, the molecular weight is not determined by the ratio of monomer to initiator. Secondly, the molecular weight distribution is wide. Since the polymer chains


134 are bifunctional, intermolecular coupling can occur which increases the molecular weight (77 - 73). If the polymerization of the N C A proceeds via the activated monomer mechanism, the initiator is not incorporated in the polymer. Therefore, this mechanism has to be avoided if the polymer shall be attached to a surface in a grafting from experiment. The activated monomer mechanism is preferred in the case of strong bases as initiators. Unfortunately, amines are strong bases too, and can initiate the N C A polymerization via the activated monomer mechanism, especially when sterically hindered. Therefore, for an efficient grafting from polymerization of N C A the surface should have primary amino groups with low steric hindrance.

ir Figure 3: Initiation and the various propagation reactions for the NCApolymerization via the activated monomer mechanism

The Initiating Layer Design. WHITESELL and coworkers suggested the use self-assembled monolayers of thiols on gold to prepare an initiating layer in which the steric hindrances are


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minimized. They prepared specially designed thiols, whose footprint on the surface exactly match the diameter of the polypeptide helix (6). We have chosen a method to prepare initiating layers with adjustable density of active sites which is not as synthetically demanding. We prepared mixed self assembled monolayers of functionalized (Br end group) and non fiinctionalized (methyl end group) undecyltrichlorosilanes in different mixing ratios and subsequently transformed the bromine groups into amino groups. Assuming that the functional groups are moleculary dispersed at the surface of the monolayer, these mixed monolayers should avoid any steric hindrances at the initiating sites, too. Silanes were chosen for self-assembling, because they form very stable monolayers which are well defined (76): The headgroups constitute a two-dimensional polysiloxane network with some attachments to the surface (77, 18). The alkyl chains form a well-ordered layer and the functional groups, if carefully selected not to compete with the headgroups in interaction with the surface, are located atop this layer (79). Preparation and characterization of the mixed self assembled monolayers. The monolayer preparation is described in detail in a previous paper (20). The thickness of the layers was measured by ellipsometry and x-ray reflectivity. The data are in accordance with a monolayer with the above-mentioned structure (20). The characterization, in terms of the monolayer composition, has been done by means of contact angle measurements and X-ray photoelectron sprectoscopy (XPS). The results are depicted in Figure 4.

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Figure 4: Advancing contact angles and Br 3d XPS intensity for the mixed self-assembled silane monolayers


136 It is clearly evident that there is a nonlinear dependence of the contact angle and the bromine XPS signal intensity on the composition of the solution. This can be explained by a preferred adsorption of the bromine terminated silane. Such preferred adsorption of slightly different molecules in the formation of self assembled monolayers has been described for thiols on gold (27). F R Y X E L L and coworkers recently reported no preferred adsorption for mixed silane monolayers (CI6), although the contact angles in this work show a strong non-linear dependence on solution composition, too (22). The bromine groups are subsequently converted into amino groups (see Figure 5) (20, 23 - 25). This in-situ modification was followed by means of contact angle and XPS measurements. The contact angle measurements clearly indicate a strong increase in hydrophilicity with the in-situ modification, whose extent depends on the density of functional groups at the surface (20). Some characteristic XPS spectra are shown in Figure 5. Br

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Figure 5: Schematic of the in-situ modification of mixed SAMs and the corresponding XPS-spectra


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After treating the monolayers with sodium azide, there is a new signal in the XPS spectrum from the nitrogen. The signal is comprised of two peaks, consistent with the structure of the azide moiety. The substitution is not complete, as indicated by the residual bromine signal. From the decrease in the bromine signal an 80% - 90% conversion can be estimated. On the other hand, the reduction of the azide moieties with lithiumaluminiumhydride seems to be complete, since there is only one nitrogen peak in the XPS spectrum of the amino-terminated SAMs. Summarizing the characterization of the monolayers and the in-situ modification, it can be concluded that we have mixed self-assembled monolayers, which are partially terminated with primary amino groups, and the concentration of these initiating sites can be adjusted via the composition of the adsorption solution. Due to a preferred adsorption and a non-complete substitution the solution composition does not match exactly with the monolayer composition.

Polymerizations The self-assembled monolayers with terminal amino groups can be used to initiate a polymerization of glutamate-NCA. The grafted polymer layer can be proven by means of FTIR spectroscopy, which gives a spectrum of pure α-helical e.g. poly-y-benzyl-Lglutamate (20). No β-sheet material can be detected, as is the case for the polymerization of methyl-L-giutamate-NCA using for example aminopropyltriethoxysilane or (4-aminobutyl)dimethylmethoxysilane layers (9). Thicknesses of the polymer layers were determined by ellipsometry and X-ray reflection. The latter method also gives the roughness of the polymer layers (20). The data obtained for a polymerization using benzyl-L-glutamate-NCA are compiled in Figure 6. The thickness of the polymer layers grafted from the self-assembled monolayer depends on the concentration of amine moieties at the surface. The thickness has a maximum for monolayers prepared from solutions with approximately 40% - 60% of functionalized silane. In addition, the roughness of the air/polymer interface (depicted as symbol size) has a minimum for this monolayer composition. Therefore, we assume that for this initiator concentration at the surface the polymerization is less interfered. This result is in accordance with the considerations made in the paragraph "Design of the Initiating Layer". The monomer for the polymerizations discussed in the previous section was prepared by the classical method (77, 20, 26) and purified by recrystallization. The polymerizations were carried out by dissolving the monomer and placing the substrates into the solution carefully excluding moisture. According to DORMAN, NCAs have a higher reactivity and yield higher molecular weights if the monomer is purified by a special procedure involving a second treatment with triphosgene (27, 28). A methyl-Lglutamate-NCA prepared according to this method and recrystallized several times in a closed apparatus in fact yielded much thicker polymer layers in a grafting from


138 experiment (see Figure 6). The polymer film is thicker for all compositions of the monolayer. The increase in thickness due to the use of the more reactive monomer is more pronounced than the thickness variation due to the differences in initiator site concentration. Therefore, the influence of the monomer purity seems to be more important than adjustment of the steric requirements.

thickness; 12 = roughness /A thickness, highly reactive monomer

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150) are, in general, not accessible using these initiators (but only by those which initiate via the activated monomer mechanism (77 - 13)), because of the nature and frequency of termination reactions. In the case of polyglutamates, cyclization of the terminal monomelic unit (see Figure 7) is assumed to be a termination reaction (73). Furthermore, in several polypeptides polymerized from NCAs, hydantoic acid units 13 have been found as the end group. The mechanism of the termination leading to this group is still under discussion (73). Impurities like free amino acid, due to an incomplete reaction or hydrolysis of NCA, may result in further side reactions which terminate the polymerization reaction. The activated monomer mechanism is less sensitive to termination reactions since the active site is located on a monomelic unit n


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and every growing chain has two sites which allow growth. Furthermore, the polymerization is much faster for this mechanism than for the amine mechanism. Therefore, the propagation is preferred over side reactions and terminations yielding higher degrees of polymerization.

Figure 7: Cyclization as a termination reaction and hydantoic acid end groups observed in the NCA-polymerization via the amine mechanism The finding that more pure and with that more reactive monomers result in a higher layer thickness is in accordance with the finding that special non solution grafting from polymerizations result in thicker polymer films. C H A N G and F R A N K reported polymer layers up to 400 Â thick obtained by a polymerization from the gas phase (or after sublimation of the monomer onto the substrate, respectively) (29). WIERINGA and SCHOUTEN obtained layer thickness in the range of 250 Â by polymerizing highly reactive benzyl- and methyl-L-glutamate-NCA by heating a spin coated monomer film to temperatures above the melting point (9, 30). In this approach the propagation is accelerated due to the high concentration of monomers and the elevated temperature.

Conclusions Self-assembled monolayers were prepared with terminal amino groups which can serve as initiators for a grafting from polymerization of N-carboxyanhydrides. The initiator site density was varied by preparing mixed self-assembled monolayers in order


140 to match the steric requirements of the growing chain and minimize the pertubations of the polymerization process. This was done by coadsorption of bromine- and methylterminated silanes and a subsequent modification including substitution of the bromine by azide and a reduction to amino groups. The bromine-terminated silanes show a preferred adsorption over the methyl-terminated ones. The substitution of the bromine by azide proceeds with approximately 80%-90% yield under the conditions applied. The partially amino terminated self assembled monolayers can be used to initiate the NCA-polymerization. The initiator site density has an influence on the polymer layer thickness. As expected, there is an optimum density of initiating sites for which the thickness has a maximum and the roughness of the air/polymer interface has a minimum. This optimum corresponds to approximately 40% -60% functionalized silanes in solution. On the other hand, the reactivity of the monomer, which can be controlled to some extent by the preparation and purification procedure, has an even bigger influence on the polymer layer thickness. The thickness variation within a series of experiments due to the different initiator site density is approximately 40%, but it is almost 100% by the change in the monomer purity. For fabrication of polypeptide layers with a thickness that exceeds the limits that have been reported in literature, it is necessary to further reduce side and termination reactions.

Acknowledgment We thank Wacker Chemitronic GmbH for the donation of silicon wafers and the Volkswagen Stiftung for financial support.

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( 13) H.R. Kricheldorf , in: Models of Biopolymers by Ring Opening Polymerization, Ed. S. Penczek, CRC-Press, Boca Raton, Fl, 1990 ( 14) Daly, W.H.; Poche, D. S.; Negulescu, I.I. Prog. Polym. Sci. 1994,19,79 ( 15) Daly, W.H.; Negulescu, I.I.; Russo, P.S.; Poche, D.S. ACS Symp. Ser. 1992, 493, 292 ( 16) Ulman, A. An Introduction to Ultrathin Organic Films - From Langmuir-Blodgett to Self-Assembly, Academic Press, San Diego 1991 and references cited therein ( 17) Tidswell, I.M.; Ocko, B.M.; Pershan, P.S.; Wasserman, S.R.; Whitesides, G.M.; Axe, J.D. Phys. Rev. B: Condens. Matter. 1990,41,1111 ( 18) Tidswell, I.M.; Rabedeau, T.A.; Pershan, P.S.; Folkers, J.P.; Baker, M.U.; Whitesides, G.M. Phys. Rev. B: Condens. Matter. 1991, 44, 10869 ( 19) Bierbaum, K.; Hähner, G.; Heid, S.; Kinzler, M.; Wöll, Ch.; Effenberger, F.; Grunze, M. Langmuir 1995, 11, 512 ( 20) Heise, Α.; Menzel, H.; Hyun Kim, Foster, M.D.; Wieringa, R.H.; Schouten, A.J.; Erb, V.; Stamm, M. Langmuir 1997, 13, 723 ( 21) Bain, C.D. ; Evall, J.; Whitesides, G.M., J. Am. Chem. Soc. 1989 111, 7155 ( 22) Fryxell, G.E.; Rieke, P.C.; Wood, L.; Engelhard, M.H.; Williford, R.E.; Graff, G.L.; Campbell, A.A.; Wiacek, R.J.; Lee, L.; Halverson, A. Langmuir 12, 5064 (1996) ( 23) Balachander, N.; Sukenik, C. N. Langmuir 1990, 6, 1621 ( 24) L.M. Lander, Ph.-D. Thesis, The University of Akron, Akron, OH (1994) ( 25) Liebmann, Α.; Lander, L.M.; Foster, M.D.; Brittain, W.J.; Vogler, E.A.; Satija, S.; Majkrzak, C.F. Langmuir 1996, 12, 2256 ( 26) Fuller, W.D.; Verlander, M.S.; Goodmann, M. Biopolymers 1976,15,1869 ( 27) Dorman, L.C.; Shiang, W.R.; Meyers, P.A. Synth. Commun. 1996, 22, 3257 ( 28) Eckert, H.; Förster, B. Angew. Chem. Int. Ed. Engl. 1987, 26, 894 ( 29) Ying-Chih Chang, C.W. Frank, following chapter this book ( 30) Wieringa, R.H.; Schouten, A.J.; Erb, V.; Stamm, M. presented at the 7th International Conference on Organized Molecular Films - LB7, 10.-15.9. 1995, Numana (Ancona), Italy


glutamates Grafted from Self-Assembled Monolayers - American

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