Bonding, Organization, and Dynamical Growth Behavior of Tripeptides

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Bonding, Organization, and Dynamical Growth Behavior of Tripeptides on a Defined Metal Surface: Tri-L-alanine and Tri-L-leucine on Cu{110} S. M. Barlow,* S. Haq, and R. Raval Leverhulme Centre for Innovative Catalysis and Surface Science Research Centre, Department of Chemistry, University of Liverpool L69 7ZD, United Kingdom Received October 16, 2000. In Final Form: February 20, 2001 The behavior of the tripeptides tri-L-alanine and tri-L-leucine, deposited under ultrahigh vacuum conditions onto a clean Cu{110} surface, has been studied using reflection-absorption infrared spectroscopy and low-energy electron diffraction. Both peptides remain intact upon landing on the Cu{110}surface and are present in their anionic form. Bonding to the surface is through the terminal carboxylate ions (COO-) and amino groups (NH2) with the CdO functionalities of the amide groups (CONH) also involved in the process. Tri-L-alanine shows a complex range of adsorption phases which are sensitive to growth conditions. At high flux and with the substrate held at room temperature (300 K), three phases are identified. Phase I occurs at low coverage with tri-L-alanine molecules randomly adsorbed and isolated from each other. As coverage increases, phase II is formed which represents a monolayer with intermolecular hydrogen bonding occurring across the surface. At higher coverages, a saturated bilayer, phase III, is created with the perpendicularly oriented CdO functionalities of the amide groups being involved in strong interlayer H-bonding. There is evidence that, locally, phase III has strong similarities to the antiparallel β-sheet form of the solid crystal although no long range ordered surface structures are seen. Multilayers are formed under high flux conditions when the Cu{110} surface is cooled to 83 K. Tri-L-leucine bonds to the surface with its longer, bulkier side chains aligned along the surface normal which sterically inhibit phase III bilayer growth. Under low flux conditions, both molecules reorient after initial adsorption so that their amide CdO functionalities are more flat lying, possibly chelating to the surface, making it difficult to grow higher coverage phases.

1. Introduction The manner in which biological molecules bind to surfaces is important for a number of applications such as biosensors, biocatalysis, and biomaterials and also for biocompatibility issues. A knowledge of the functionalities involved in bonding to the surface, the molecular orientation with respect to the surface, and the molecular conformation adopted can yield an understanding of the possible ways in which a molecule can interact with the surrounding medium. However, fundamental studies of the adsorption of biological molecules to surfaces, especially clean, defined surfaces under controlled conditions, have been very limited. A number of studies of simple amino acids which have been vacuum deposited onto clean well-defined metal surfaces have been undertaken,1-3 but studies for larger molecules, such as peptides and proteins, have been confined to films created from solution onto polycrystalline metal surfaces or colloidal metal particles.4-8 Under these latter conditions, it is not possible to finely control parameters such as the adsorption rate or film thickness or to consider effects due to adsorption (1) Williams, J.; Haq, S.; Raval, R. Surf. Sci. 1996, 368, 303. (2) Barlow, S. M.; Kitching, K. J.; Haq, S.; Richardson, N. V. Surf. Sci. 1998, 401, 322. (3) Lofgren, P.; Krozer, A.; Lausma, J.; Kasemo, B. Surf. Sci. 1997, 370, 277. (4) Lee, H. L.; Suh, S. W.; Kim, M. Y. J. Raman Spectrosc. 1988, 19, 491. (5) Lee, H. L.; Kim, M. S.; Suh, S. W. Bull. Korean Chem. Soc. 1988, 9, 218. (6) Herne, T. M.; Aherne, A. M.; Garrell, R. L. J. Am. Chem. Soc. 1991, 113, 846. (7) Herne, T. M.; Aherne, A. M.; Garrell, R. L. Anal. Chim. Acta 1991, 246, 75. (8) Liedberg, B.; Ivarsson, B.; Lundstrom, I.; Salaneck, W. R. Prog. Colloid Polym. Sci. 1985, 70, 67.

at specific sites. Therefore, in the present work we have sought to extend our previous studies for simple amino acids1,2 on the ordered Cu{110} surface to tripeptides which we have vacuum deposited under ultrahigh vacuum (UHV) conditions onto the same surface. There is evidence that even short peptide segments such as tripeptides can exhibit topological characteristics associated with much larger proteins,9 so studying tripeptides can be viewed not only as extending amino acid work but also as providing models for the interactions associated with much larger biomolecules. Copper was chosen as a biologically interesting surface known to complex with a range of peptides in solution. A tripeptide essentially consists of three amino acid molecules (H2N-CH(R)-CO2H) joined together with the elimination of water and the formation of amide bonds. In this study, the tripeptides tri-L-alanine, where R ) CH3, and tri-L-leucine, where R ) CH2CH(CH3)2, as shown in Figure 1a,b, were studied. Both tripeptides are solid white powders at room temperature and, as is also the case for amino acids, are zwitterionic in the solid form. These tripeptides were chosen as representatives of poly(leucine) and poly(alanine) which are known to catalyze oxidation reactions, for example, the enantioselective epoxidation of certain ketones and dienones. Of particular interest is the fact that it is suspected that the active site may involve only a smaller peptide unit.10 In this paper, we report results of the adsorption and growth of tri-L-alanine and tri-L-leucine on Cu{110} using the technique of reflection-absorption infrared spectroscopy (RAIRS) in order to determine the nature of the (9) Go, K.; Parthasarathy, R. Biopolymers 1995, 36, 607. (10) Lasterra Sanchez, M. E.; Roberts Stanley, M. J. Chem. Soc., Perkin Trans. 1995, 1, 1467.

10.1021/la001441z CCC: $20.00 © 2001 American Chemical Society Published on Web 04/24/2001

Behavior of Tripeptides on a Defined Metal Surface

Figure 1. (a) Tri-L-alanine. (b) Tri-L-leucine.

adsorbed species and the functionalities involved in the bonding to the surface and to yield information regarding the molecular conformation. Furthermore, for RAIRS, vibrations which give rise to spectral features are confined to those where the dipole moment of the adsorbed molecule has a component perpendicular to the metal surface enabling the molecular orientation to be deduced. We note, however, that information regarding absolute orientation can be difficult to obtain for large molecules such as tripeptides which have little symmetry and a large number of possible vibrations with much intermixing of modes. The RAIRS technique is, nevertheless, a powerful probe of changes in orientation of the tripeptide in response to changing conditions, for example, coverage and temperature. In addition, the surface structure of the tripeptides on the Cu{110} was also investigated using low-energy electron diffraction (LEED), to probe the existence of longrange order. 2. Experimental Section The Cu{110} crystal was mounted in a UHV chamber of base pressure ∼1 × 10-10 mbar and cleaned by cycles of Ar+ bombardment and annealing to ∼900 K. LEED was used to confirm the sharp (1 × 1) pattern characteristic of clean Cu{110}. The tripeptide powder was contained in a small, electrically heated glass tube, separated from the main vacuum chamber by a gate valve and differentially pumped by a turbomolecular pump. Before evaporation, the tripeptide was outgassed for several hours at ∼350 K with the gate valve closed. The vacuum chamber contained a quadrupole mass spectrometer which enabled the mass fragments of the tripeptide present to be monitored during deposition. The tri-L-alanine was found to evaporate from about 407 K with the main chamber pressure rising to around 1 × 10-8 mbar. The tri-L-leucine evaporated at a slightly higher temperature from around 421 K. The RAIR spectra were measured using a Mattson-Galaxy FT-IR spectrometer interfaced to the UHV chamber with KBr optics and windows. The infrared radiation was detected with a liquid nitrogen cooled HgCdTe detector which had a spectral range of 650-4000 cm-1. The spectrometer was operated with a resolution of 4 cm-1, and typically 400 scans were taken resulting in collection of a full spectrum in about 2 min. At the start of each experiment, a spectrum of the clean metal surface was taken to give a background reference R0. Subsequent spectra were recorded in real time and presented as the ratio of the difference to the background spectrum (R - R0)/R0.

3. Results and Discussion 3.1. Framework for Analysis. To aid discussion of the data, it is important to outline some general issues regarding peptides, including their conformation, vibra-

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tional spectra, and interactions with metal surfaces, which critically affect our analyses. (a) Peptide Conformation. The conformation adopted by a peptide crucially determines its activity and performance, and a number of different structures have been found, with H-bonding being a significant feature in all of the conformations and also prevalent in various intermolecular interactions. Typical conformations are R-helices (with the amide bonds forming steps of a helicoidal staircase), β-sheets (with either parallel or antiparallel rows of molecules which are pleated or folded), and assorted turn structures.11,12 The exact sequence of the amino acid within a peptide also affects the conformation. For example, tripeptides formed from the constituent amino acids glycine (Gly), alanine (Ala), and/or leucine (Leu) exhibit β-sheet conformations for the sequences GlyGlyGly, AlaAlaAla, or LeuLeuLeu but an R-helical conformation for the sequence GlyAlaLeu.9 Infrared analyses of peptides, polypeptides, and proteins show that although the main features observed are similar to those of the constituent amino acids, the additional bands derived from the amide bonds are useful diagnostic tools for conformational analysis.11-13 These are generally identified according to the classification system Amide I, Amide II, Amide III, and so forth with each band being a combination of vibrations resulting from the amide resonance. For interpretation of peptide spectra, the most useful vibrations are Amide I at ∼1650 cm-1 (which is mainly due to the CdO stretch of the amide bond) and Amide II at ∼1540 cm-1 (which arises largely from the NH deformation of the amide bond). Normal coordinate analyses, comparing vibrational spectra of various peptide turn structures, have shown that the Amide I band, in particular, is very sensitive to conformation. A general observation appears to be that the amide bands tend to occur at a slightly higher wavenumber for folded R-forms than for extended β-forms. Typically, the Amide I and II bands for an R-helical form occur around 1650-1660 and 1540-1550 cm-1 but occur around 1630 and 1530-1525 cm-1, respectively, for the β-sheets. However, despite the general success of such analyses, we note that it cannot be expected that a structure can be predicted from infrared data alone. Amide bands are also often split owing to the different environments of the various amide groups in any particular molecule or crystal which give rise to a range of coupled vibrations. For example, randomly arranged molecules of polypeptides also have Amide I and II bands around 1656 and 1535 cm-1, which further complicates the interpretation of spectra unless some prior knowledge exists regarding the expected conformation. (b) Peptide-Metal Interactions. An important aspect of peptide/metal interfaces is the nature of the bonding between them. Both amino acids and peptides have been found to bind to metal surfaces in a manner analogous to the formation of organometallic complexes in solution. For example, glycine and alanine deposited by direct evaporation or adsorption from solution onto metal surfaces such as copper show infrared spectra that can be compared directly to those from the equivalent metalamino acid complexes.1,2,14 From a consideration of the reactions of metal ions in solutions of peptides, it is known that the most common site for coordination of a peptide (11) Avram, M.; Mateescu, Gh. D. Amino acids and Proteins. In IR Spectroscopy; Wiley-Interscience: New York, 1972; Chapter 11. (12) Bandekar, J. Biophys. Acta 1992, 1120, 123. (13) Bellamy, L. J. The Infrared Spectra of Complex Molecules, 2nd ed.; Chapman & Hall: London, 1980; Chapter 12. (14) Ihs, A.; Liedberg, B.; Udval, K.; Tornkvist, C.; Bodo, P.; Lundstrom, I. J. Colloid Interface Sci. 1990, 140, 192.

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to a metal is the terminal amino NH2 group.15,16 This usually coordinates in conjunction with the O atom of the amide group to form a five- or six-membered chelate ring. The O atom of the amide group will not bond with the metal if such a chelate structure is not formed. The protonated N of the amide bond is never involved in bonding to the metal as this would imply a tetrahedral configuration which is energetically and geometrically unfavorable.16 However, the neutral or deprotonated N atom of the amide bond can be involved in bonding to the metal and gives rise to a large range of metal-peptide species.15,16 Deprotonation of the amide group, and thus opportunities for forming complexes, can be promoted in the presence of the metal, when the peptide is in its anionic form with terminal COO- and amino NH2 groups. Turning to peptide adsorption on metal surfaces, most of the published literature relates to dipeptides or tripeptides on colloidal silver studied by SERS (surfaceenhanced Raman spectroscopy). The favored binding site is thought to be the N terminal residue with the COOterminal and the amide group involved in some but not all cases, depending on the peptide.4-7 Interestingly, there is some evidence that large molecules containing peptides can undergo structural changes on adsorption. Liedberg et al.8 have considered the RAIR spectra of proteins adsorbed from solution on a number of metal films and report that on adsorption of the protein, the Amide I band shifts to a higher frequency, typically from 1649 to 1670 cm-1 for fibrinogen and from 1635 to 1666 cm -1 for β-lactoglobulin. They observe that the shift is greater for proteins with an initial β-sheet structure than for those with an R-helix or disordered structure and comment that this possibly reflects a change in the structure of the protein on adsorption. A final and very important point to note is that upon adsorption on a metal surface, the absolute frequency of any particular mode of vibration of the adsorbed peptide will not just depend on the conformation of the molecule. Mechanical coupling between the metal surface and the peptide together with chemical coupling due to charge transfer from the metal to the ligand and dipole coupling between adsorbed molecules will all affect the observed vibrations of the adsorbed peptide molecule, leading to the possibility of significant shifts.1,2 Therefore, the molecular conformation of the adsorbed molecule cannot be expected to be deduced from the frequency of the amide vibrations alone in the manner that is often employed for standard peptide analyses.11-13 3.2. Tri-L-alanine. Tri-L-alanine exists in two main crystalline forms, namely, as parallel (when in an unhydrated form) or, more commonly, as antiparallel (when in a hemihydrate form) β-pleated sheets.17,18 In each case, a network of H-bonds connecting the amide and terminal groups and water (if applicable) forms sheets of molecules which then assemble into a 3-D structure. The two forms are shown in Figure 2a,b, and it can be seen that there are more opportunities for H-bonding between the antiparallel molecules than for the parallel rows of molecules. Trialanine has been the subject of a number of infrared studies both in crystalline and solution forms, and the main vibrations are summarized in Table 1,19-21 as an aid (15) Freeman, H. C. Inorg. Biochem. 1973, 1, 121. (16) Freeman, H. C. Adv. Protein Chem. 1967, 22, 257. (17) Hempel, A.; Camerman, N.; Camerman, A. Biopolymers 1991, 31, 187. (18) Fawcett, J. K.; Camerman, N.; Camerman, A. Acta Crystallogr. 1975, B31, 658. (19) Ellenbogen, E. J. Am. Chem. Soc. 1956, 78, 363. (20) Blanchard, S. J. Mol. Struct. 1977, 38, 51. (21) Qian, W.; Bandekar, J.; Krimm, S. Biopolymers 1991, 31, 193.

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Figure 2. (a) Parallel β-sheets of tri-L-alanine. (b) Antiparallel β-sheets of tri-L-alanine.

to subsequent analysis. Typically, for such complex molecules the normal mode of vibration usually involves amplitude in a number of motions, involving a number of functional groups, so an assignment indicates the major vibration contributing to the peak. A detailed study21 has been made of the parallel and antiparallel β-sheet forms which importantly shows that although the positions of the infrared bands are similar in both forms, their relative intensities and breadths vary significantly with conformation. For example, the parallel form has sharper Amide I bands than the antiparallel form, which can be explained by the fewer opportunities for H-bonding present in the former configuration. The coordination and structure of a range of peptides with copper in solution have been extensively studied,22-24 and the known copper trialanine complex22 where a chelate structure with five-membered rings is created, Figure 3, provides a starting point for determining bonding sites at the extended Cu{110} surface. In the complex, the copper coordinates to the terminal NH2, both deprotonated amide N atoms and the terminal COO- group. A number of intermediate structures are also possible22,24 where the copper coordinates to either (i) the terminal NH2 and adjacent amide CdO or (ii) the terminal NH2, the adjacent deprotonated amide N, and the further amide CdO before giving the structure seen in Figure 3. Unfortunately, no IR data are available for this Cu-tri-L-alanine complex. However, previous RAIRS, LEED, and XPS (X-ray photoelectron spectroscopy) studies1 of the amino acid Lalanine on Cu{110} can be used to aid our analysis. L-Alanine was shown to adsorb in an anionic form with bonding to the surface via the terminal carboxylate (COO-) and amino (NH2) groups. Table 1 includes the vibrational assignments for this Cu/L-alanine system. 3.3. Adsorption of Tri-L-alanine on Cu{110}. The most interesting aspect of the RAIR spectra obtained for tri-L-alanine adsorbed on the Cu{110} surface is that they show variations with coverage, temperature, and flux, (22) Owens, G. D.; Phillips, D. A.; Czarnecki, J. J.; Raycheba, J. M. T.; Margerum, D. W. Inorg. Chem. 1984, 23, 1345. (23) Kroneck, P. M. H.; Vortisch, V.; Hemmermich, P. Eur. J. Biochem. 1980, 109, 603. (24) Sigel, H.; Martin, R. B. Chem. Rev. 1982, 82, 385.


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Table 1. Infrared Vibrations for Trialaninea frequency cm-1

assignment NH str (NH3+) CH as/s str 2×CH3 def Amide I

tri-L-alanine (in Nujol) (ref 19)

zwitterionic trialanine in solution (ref 20)

crystalline tri-L-alanine (ref 21) //l-chain β

3311 3030 2045 2975 2925

3342 s 3282 s

2984 mw

2980 mw

1667

1668

1637

1645

1686 sh 1668 w 1649 vs

NH2 def/r COO- as str CN str/NH def Amide II

1624 1595 1530

1590

1628 vw 1593 vs

CH as def

1490 1445

COO- s str

1470 1404

1475 1460 1448 1408

CH3 s def CH def/COO- s str NH2 r/def/NC str

1274

NC/CC str NH2 w/COO-s str CH3 def/r

anti-//l-β

3357 vs

1550 sh 1525 s 1451 ms 1422 sh

1691 mw 1667 sh 1647 sh 1641 vs 1623 w 1595 mw

1407 s

1547 1536 s 1459 ms 1447 w 1443 vw 1411 s

1393 sh 1382 w 1363 m

1400 sh 1377 mw 1364 ms

1279 m

on Cu{110}at 300 K (ref 1)

low coverage

high coverage

2935 2876

2985 2931 2876

1576 1613

1576 1630

1466

1462

1411

1415

1373

1377

1302 1276

1289 1271

1145

1167

1086 1036 1011

1086 1036

919

919

1268 m 1234 ms

1204 ms 1154 m 1128 m

1148 ms

CCN as str CN str/COO- s str/CH3 r CH3 def CNC def CN str/COO- s def

L-alanine

1081 mw 1062 m

1055 ms

1000

Key: s ) symmetric, as ) asymmetric, str ) stretch, def ) deformation, r ) rock, vs ) very strong, s ) strong, m ) medium, w ) weak, sh ) shoulder. a

Figure 3. Copper/tri-L-alanine complex.

particularly with respect to relative intensities and breadths of the absorption peaks. This behavior suggests that the interface is particularly sensitive to adsorption parameters and that a number of surface phases are created under different conditions. In fact, we find that under high flux conditions, three adsorption phases, plus the multilayer, can be identified as shown in the schematic adsorption phase diagram of Figure 4. The RAIR spectra associated with each of the phases I-III which are formed at room temperature are shown in Figure 5, and those

Figure 4. Adsorption phase diagram of tri-L-alanine/Cu{110} under high flux conditions.

produced from the phases grown at 83 K, including the multilayer, are shown in Figure 6. The vibrational assignments deduced for these phases are shown in Table 2. A detailed description of the phases is given below. Phase I. This is a low coverage phase and is observed to occur upon adsorption at both 300 and 83 K. The absorption peaks are relatively sharp and occur at comparable wavenumbers to those of the RAIR spectra of the L-alanine/Cu{110} system, Table 1, with additional

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Figure 5. RAIR spectra of tri-L-alanine deposited on Cu{110} at 300 K under high flux conditions.

Figure 6. RAIR spectra of tri-L-alanine deposited on Cu{110} at 83 K under high flux conditions.

peaks due to the Amide I and II vibrations being identifiable by comparison with the infrared spectra of tri-L-alanine, Table 1. The main absorption peaks observed are tabulated in Table 2 and are due to the vibrations associated with the symmetric and asymmetric carboxylate stretches around 1400 and 1615 cm-1, respectively, the Amide I (principally CdO stretch) and Amide II (principally NH deformation) bands around 1672/1640 and 1522 cm-1, respectively, the CH deformation around 1468 cm-1, and the CH stretches around 2979 cm-1. The presence of all the expected vibrations for tri-L-alanine infers that the tripeptide is adsorbed intact. In addition, the existence of vibrations associated with the terminal COO- and NH2 groups indicates that the tri-L-alanine bonds to the Cu{110} surface in its anionic form in a manner similar to that seen for L-alanine. The existence of the Amide I vibrations, together with those of the terminal groups, suggests that at least one of the O atoms of the two amide groups interacts with the surface in an manner analogous to that of intermediate tri-Lalanine/Cu organometallic complexes. However, we are not able to ascertain from the spectra whether the N atom of the amide group deprotonates and also bonds to the surface. The Amide II vibration (NH def) is relatively weak, suggesting some deprotonation, but this vibration could also have been weak as a result of the direction of the

dipole moment of this functionality with respect to the surface and this is the more likely situation. An annealed version of phase I can also be created by warming higher coverage phases to around 473 K before complete desorption of the tri-L-alanine from the surface above 513 K. For the annealed phase I, the main Amide I vibration occurs at a higher frequency than on initial deposition (1696 cm-1 compared to 1676 cm-1). Normal coordinate calculations21 on the infrared spectra of crystalline trialanine (both parallel and antiparallel arrangements) have identified that the Amide I vibrations are a combination of vibrations due to the two amide CdO groups in the molecule. The vibrations of each CdO group vary according to the local environment; that is, there are slight frequency differences between the vibrations associated with the CdO groups near the terminal NH2 and those nearer the terminal COO- group with the overall Amide I group frequency being a combination of the two CdO vibrations. It appears that the contributions are such that the Amide I vibration for the crystalline form, Table 1, consists broadly of a vibration at ∼1680 cm-1 due to the CdO group near the NH2 terminal, a vibration at ∼1665 cm-1 due to the CdO group near the COO- terminal, and a broader vibration around ∼1640-1650 cm-1 due to both CdO groups. Thus, a higher frequency Amide I vibration


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Table 2. RAIR Spectra Peak Assignments of Tri-L-alanine on Cu{110} Surface (a) Phases Grown at 300 K under High Flux Conditions frequency cm-1 assignment CH3 as str CH3 s str Amide I COO- as str NH2 def Amide II CH3 as def COO- s str CH3 s def NH2 w/COO- s str CCN as str

annealed II (warmed to 388 K)

annealed I (warmed to 473 K)

2987 2948 1676 1636 1618

2974 2938 1688 1628

2976 2939 1696 1624 1599

1539 1455 1407

1539 1456 1408

1534 1454 1407

1534 1456 1408

1162 1088

1163 1088

1157 1088

phase I

phase II

phase III

2979 2937 1678 1640 1615 1568 1522 1458 1398 1368 1154 1086

2980 2939 1672 1627 1621

(b) Phases Grown with the Substrate at 83 K under High Flux Conditions frequency cm-1 assignment CH3 as str CH3 s str Amide I COO- as str NH2 def Amide II CH3 as def COO- s str CH3 s def NH2 w/COO- s str CCN as str

phase I

1669

annealed III (warmed to 303 K)

annealed II (warmed to 373 K)

2979 2939 1671

2979 2938 1677 1636

2979 2939 1688 1655/1638

1558 1540 1456 1404

1560 1541 1458 1405

1561 1543 1458 1410

1163

1161

multilayer

1622/1593 1522 1452 1404 1375 1164 1091

1094

on annealing could reflect a change in bonding/orientation of the CdO group at the end of the molecule nearer the NH2 terminal. However, as we discussed earlier, the absolute values of vibrations will change on adsorption, so such a conclusion is necessarily speculative. The conformational complexity of the tri-L-alanine molecule makes it very difficult to use the dipole metal surface selection rule to determine unambiguous orientational information. Most of the vibrations associated with the crystal, Table 1, are represented in the RAIR spectra, Figures 5 and 6 and Table 2, and the molecule has little symmetry. However, some general considerations can still be made. For example, the peaks due to the Amide I vibrations are clearly resolved when the tri-L-alanine is adsorbed on the copper surface indicating, from the metal surface selection rule, that the amide groups are oriented with their CdO functionalities mainly perpendicular to the surface. Because the usual conformation of tri-Lalanine is such that one CdO group is on either side of the main planar backbone of the molecule, the arrangement indicated by the RAIR data is for one CdO to be directed away from the surface and the other to be directed toward the surface, allowing a possible bonding interaction. Within phase I, the exact intensity profiles of the spectra vary under essentially the same adsorption conditions. We attribute this to a somewhat disordered phase with molecules randomly sticking on available sites on the Cu{110} surface. However, all these spectra are relatively sharp, especially in the Amide I region, with little evidence of H-bonding, indicating a low level of interaction between the tri-L-alanine molecules on the surface. Therefore, phase I may be envisaged as a disordered “sea” of individual and isolated adsorbed molecules, created when adsorption is first initiated. Despite the general lack of two-dimensional order, this is a kinetically favored phase

as it is readily observed on initial deposition and is also recreated by warming higher coverage phases. Phase II. As coverage of tri-L-alanine is increased at room temperature, the main feature observed in the RAIR spectrum is an intense, broad peak around 1676 cm-1 containing the Amide I vibrations, asymmetric carboxylate stretch, and the NH2 deformation, Table 2 and Figure 5. There are still some differences in relative intensities and breadths of spectra recorded on different occasions, but the general features of the phase are reproducible. Phase II is not readily observed when coverage is increased at 83 K because the multilayer grows very rapidly. However, an annealed version of this phase can be created by warming higher coverage phases, including the multilayer, to around 373 K, Table 2 and Figure 6. Again, as with the annealed phase I, the basic spectrum typical of the phase is created but there is an upward shift in the frequency of the Amide I of this annealed phase II, indicating possible changes in the bonding interaction with the surface. In phase II, the absorption peaks become broader, especially for those peaks associated with the Amide I bands (mainly CdO stretch). This behavior is strongly indicative of intermolecular H-bonding occurring (e.g., in crystalline form) between adjacent tri-L-alanine molecules. This intermediate coverage phase is built from the randomly adsorbed phase so the possibility for intermolecular interactions occurs both across the surface as vacant sites are occupied and above the surface as “second layer” molecules arrive. This intermediate coverage phase II appears to be a transition into the high coverage phase III and can be attributed to monolayer coverage in which increasing lateral interactions between the adjacent molecules on the surface lead to the general breadth of the Amide I features. Further justification for this interpretation is provided by the behavior of the tri-Lleucine, which will be discussed later, where the longer

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Figure 7. Possible arrangements of H-bonded tripeptide chains at surface.

side chains of the molecule deter growth past the monolayer regime. Phase III. This phase is readily and reproducibly observed with increasing coverage for all room temperature depositions under high flux conditions and is characteristic of the fully covered surface. The spectrum is dominated by a strong broad peak around 1676 cm-1 typical of the Amide I vibration together with other Amide I vibrations around 1636 cm-1 and a peak associated with the asymmetric COO- stretch around 1618 cm-1. Other easily identifiable peaks include the CH deformation around 1456 cm-1 and the symmetric COO- stretch at 1408 cm-1, Table 2 and Figure 5. The presence of these peaks confirm that the tri-L-alanine continues to bond to the Cu{110} through its terminal COO- group and probably the terminal NH2 group and that the CdO functionality of the amide group retains a perpendicular geometry with respect to the surface with the downwardpointing CdO group possibly also involved in the bonding interaction. The most interesting aspect of this saturated phase III is that the general shape of the RAIR spectrum bears strong resemblances to that published for the antiparallel pleated β-sheets of the crystalline solid, especially in the Amide I region.21 As discussed earlier, surface coupling effects mean that the vibrational frequencies alone cannot be used to determine local organization of the molecule at the surface, but used in conjunction with the overall shape of the spectra such information may be strongly inferred. We therefore suggest that pairs of molecules are held together in an antiparallel arrangement with strong H-bonds (CdO‚‚‚HN) between the adjacent CdO and NH functionalities of the amide groups of neighboring molecules. These pairs of molecules are then H-bonded through their terminal groups to adjacent molecule pairs to form chains of tri-L-alanine molecules across the surface. As in the crystal, the strongest H-bonding occurs between the amide CdO group of one molecule and the amide HN group of an adjacent molecule. In principle, this H-bonding could be expected to take place either across the surface with the interacting CdO groups essentially parallel to the surface or away from the surface with the interacting CdO groups directed vertically upward. Figure 7 illustrates the way the pairs of tripeptide molecules could form at the surface and the direction of the Amide I vibration for each case. Application of the metal surface selection rule dictates that the amide CdO vibrations are only observed in the RAIR spectra if these groups are broadly perpendicular to the metal surface. On this basis,

Figure 8. Tri-L-alanine low flux growth at 300 K.

the configuration of Figure 7(i) would give rise to measurable CdO vibrations whereas the arrangements in Figure 7(ii),(iii) would not. Thus, it would appear that, at full coverage, the tri-L-alanine molecules are stacking vertically in essentially a bilayer arrangement, as shown in Figure 7(i), with strong similarities to the antiparallel β-sheet form of the solid crystal. No LEED patterns are seen for the Cu/tri-L-alanine system at any coverage, indicating that long-range ordered surface structures are not formed. Multilayer. This is created only when the substrate is held below room temperature and is readily observed with the Cu{110} held at 83 K. It is characterized by a large dominant Amide I band in the RAIR spectrum which grows rapidly with increasing exposure to tri-L-alanine, Table 1 and Figure 6. The multilayer appears to grow directly from the low coverage phase I, but as it forms very readily, we note that it is not easy to observe the details of any intermediate phases. The very large broad Amide I band is indicative of extensive H-bonding occurring between the molecules as would be expected for a thick layer of tri-L-alanine molecules. When the multilayer is warmed, tri-L-alanine starts to desorb from 180 K and by 300 K the RAIR spectrum becomes very similar to that obtained when adsorbing to saturation on a room temperature surface; that is, phase III is formed. On further warming to 373 K, phase II is also observed, indicating that the room temperature deposition phases are kinetically preferred. Low Flux Deposition Conditions. The phases discussed so far all occur under “high flux” conditions; that is, a constant flux of tri-L-alanine molecules is introduced over a period of time sufficient to allow continuous growth of the tripeptide film. However, if the flux of tri-L-alanine is reduced an interesting variation in the growth of the room temperature phases is seen. Phase I starts to grow as usual but it becomes difficult or impossible to grow any other phases; that is, the surface essentially becomes “nonstick”! This behavior is also observed if phase I is created and then adsorption is stopped to allow the phase to relax. This effect can best be monitored by the Amide I band at ∼1670 cm-1 and is illustrated in Figure 8. When the supply of tri-L-alanine is closed off before phase II is formed, this Amide I band, which has been growing slowly, starts to decay without material appearing to leave the surface. This variation in intensity of the main Amide I


Langmuir, Vol. 17, No. 11, 2001 3299

Table 3. Vibrational Assignments for Leucine frequency cm-1 DL-leucine

(ref 26)

Cu-(DL-leucine)2 (ref 26)

CH/CH2/CH3 str Cd0 str peptide band

2871-2962

2871-2978

COO- str NH3+ deg def NH2 sciss NH3+ s def CH3 deg def CH2 sciss COO- s str CH3 s def C-O str CH b/CH2 w, tw CH3 r/NH2 w, tw/CC str/CCCN str

1584 1612

assignment

a

L-leucine

L-leucine

(ref 25)

in Ar matrix (ref 27)

leucine-based peptide (refs 25 and 28)

2858-2975 1768 1680a 1676b

1640

1577 1608

1573 1510 1469 1442 1413 1392

1468 1454 1398 1382

1239-1361 925-1184

1251-1369 907-1204

1640 1510 1473 1453 1402 1370-1386 1285

Glycine-leucine-based peptide. b Leucyl-valine peptide.

peak indicates that the amide CdO groups associated with this frequency are changing orientation with respect to the surface and are becoming more flat lying with their dipole moment no longer perpendicular to the surface. On the introduction of more tripeptide, the band starts to slowly grow again but it is extremely difficult to grow higher coverage phases. This behavior suggests that orientational changes that occur when the adlayer is allowed to relax (either in low flux conditions or by halting adsorption) effectively block further tripeptide growth. Because this flat-lying orientation is not the initial phase at room temperature, there must be some kinetic barrier to its creation. A possible interpretation is that under low flux conditions the tri-L-alanine molecule fully chelates to the metal surface, in the same manner as the organometallic complex, Figure 3, deprotonating the amide NH group. Saturation occurs when this chelated form fully covers the metal surface. After chelation has occurred, it is difficult for the molecule to revert to its original adsorbed phase I form in which vertically pointing CdO groups provide a “docking” interaction, via H-bonding, for second layer molecules to stick to the surface. In conclusion, the important point to note regarding the organization of the tri-L-alanine at the copper surface is that the process is driven by H-bonding between the molecules. Opportunities for H-bonding determine the eventual growth mode and short range organization of the tri-L-alanine film. 3.4. Tri-L-leucine. Trileucine, like trialanine, also prefers a β-sheet rather than a helicoidal conformation in the crystalline state but adopts a slightly unusual twisted β-sheet conformation, more usually seen in larger protein molecules.9 Again, the molecules in the crystal are held together by an extensive network of H-bonds. Infrared spectra of trileucine are not available, but it can be expected that the main differences from trialanine will arise because of the larger side chain structure of the trileucine molecule. In addition, infrared spectra have been published for L-leucine,25 DL-leucine and its copper complex,26 and matrix isolated leucine in the acid form,27 and some spectral assignments are also available for glycineleucine28 and leucine-valine peptides.25 The main features of these spectra which can be used to aid in the interpretation of the tri-L-leucine on Cu{110} RAIR spectra are summarized in Table 3. (25) Larsson, L. Acta Chem. Scand. 1950, 4, 27. (26) Jackovitz, J. F.; Walter, J. L. Spectrochim. Acta 1966, 22, 1393. (27) Sheina, G. et al. Zh. Fiz. Khim. 1988, 62, 985. (28) Blout, E. R.; Linsley, S. G. J. Am. Chem. Soc. 1951, 74, 1946.

Table 4. RAIR Spectra of Tri-L-leucine on Cu{110} Grown at 300 K frequency cm-1

assignment CH3/CH2/CH str Amide I COO- as str NH2 def Amide II CH3 as def COO- s str NH2 w/COO- s str

low coverage phase I 2968 2937 2877 1671 1642 1605 1551 1468 1391

saturated coverage phase II

annealed phase

2968 2937 2877 1665

2968 2937 2877

1611 1557 1534 1468 1391 1175

1617 (broad) 1468 1396 1175

Tri-L-leucine is known to form a coordination compound with Cu2+ in solution in a manner similar to that of trialanine.22 The fully chelated complex consists of the copper ion bonded to the terminal NH2 and COO- groups and the deprotonated N atoms of the amide, but no vibrational data are available. 3.5. Adsorption of Tri-L-leucine on Cu{110}. Adsorption of tri-L-leucine on Cu{110} was conducted with the substrate held at room temperature (∼300 K). Under high flux conditions, two phases are identified corresponding to low and saturated coverage. The RAIR spectra associated with these phases are plotted in Figure 9 on the same intensity scale as Figure 6 which depicts the Cu/trialanine system. It can be seen that the low and saturated coverage phases resemble phases I and II of the Cu/trialanine system. The main features, summarized in Table 4, can be assigned to vibrations arising from the constituent amino acid, leucine, together with the Amide I and II vibrations, indicating that the tri-L-leucine is adsorbed intact. With increasing coverage, the region around the Amide I vibrations again grows into a broad, intense peak enveloping other vibrations in the 15001700 cm-1 region of the spectrum and indicating that the CdO of the amide group is oriented broadly perpendicular to the surface. The main differences from the tri-L-alanine/ Cu spectra are associated with the CH stretches around 2900 cm-1 which are much more intense and are comparable in intensity to the main Amide I peaks. These vibrations arise from the larger side chains of the tri-Lleucine, and their significant intensity suggests that they are strongly oriented perpendicular to the surface.

3300


Barlow et al.

Figure 9. RAIR spectra of tri-L-leucine deposited on Cu{110} at 300 K under high flux conditions.

On initial adsorption, a sharp peak around 1670 cm-1 (Amide I) is observed, Figure 9, indicating a low concentration of H-bonding interactions. We conclude that this phase is essentially a low coverage disordered phase I, with little interaction occurring between the molecules. With increasing coverage, this Amide I peak broadens and includes intensity due to the other Amide I vibration (∼1640 cm-1), the asymmetric stretch of the terminal COO- group, the terminal NH2 group deformation, and the Amide II vibration. The increased breadth of the Amide I peak is again indicative of increased H-bonding between adjacent molecules, and the RAIR spectrum is broadly similar to that of phase II of the tri-L-alanine system. We therefore propose that this is characteristic of monolayer coverage. Interestingly, further adsorption does not occur with phase II representing the saturated surface and a phase characteristic of the bilayer not forming. This can be understood if the effect of the longer side chains of the tri-L-leucine molecule is considered. These bulky groups are directed normal to the surface, effectively hindering the CdO functionalities of the amide groups from Hbonding to further incoming molecules, an interaction which is essential for nucleating bilayer and multilayer growth. Further evidence that this saturated phase is a monolayer is given by its behavior on warming when only one phase is created. The Amide I peak at 1670 cm-1 is seen to disappear by 400 K leaving a broad peak around 1611 cm-1 with the sharper, more intense peak around the COO- symmetric stretch region remaining. The peaks associated with the CH stretches reduce slightly in intensity as this warming occurs but remain as a feature comparable in intensity to the COO- symmetric stretch. This suggests that the molecule reorients itself on annealing with the amide CdO group becoming more flat

lying. This annealed phase is present up to a temperature of 253 K with the tri-L-leucine desorbing entirely by 473 K. 4. Conclusions Both tri-L-alanine and tri-L-leucine have been successfully evaporated under UHV conditions onto the Cu{110} surface. The tripeptides adsorb intact in an anionic form with the terminal acid group ionized as a carboxylate ion. RAIR spectra show bands typical of the constituent amino acids as well as the amide groups linking these amino acids. In addition, tri-L-leucine shows strong bands associated with the longer side chains of this molecule. Molecular orientation and organization at the surface is found to be dependent on coverage, temperature, and flux, and a number of growth phases have been identified. For tri-L-alanine, under high flux conditions, three distinct phases, I-III, are found at room temperature, characteristic of a randomly oriented disordered phase, a monolayer, and a bilayer, respectively. Although no longrange ordered surface structures are observed with LEED, the saturated bilayer has strong similarities to an antiparallel β-sheet conformation characteristic of the solid trialanine crystal. Under low flux conditions, the CdO of the amide group changes from being broadly perpendicular to the surface to a more flat-lying orientation, possibly involving chelation, which blocks further tripeptide growth. In contrast to trialanine, the longer side chains of the tri-L-leucine prevent film growth past phase II, the monolayer at room temperature. Under all conditions, the main driving force for creating the tripeptide structures at the surface appears to be opportunities for H-bonding. LA001441Z

Bonding, Organization, and Dynamical Growth Behavior of Tripeptides

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