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Surface Science 599 (2005) 207–220 www.elsevier.com/locate/susc Investigation of molecular structures and adsorption mechanisms of phosphonodipeptides by surface-enhanced Raman, Raman, and infrared spectroscopies E. Podstawka a,* , R. Borszowska b, M. Grabowska b, M. Dra˛g c, P. Kafarski c, L.M. Proniewicz b a c Laser Raman Laboratory, Regional Laboratory of Physicochemical Analysis and Structural Research, Jagiellonian University, ul. Ingardena 3, 30-060 Krakow, Poland b Chemical Physics Division, Faculty of Chemistry, Jagiellonian University, ul. Ingardena 3, 30-060 Krakow, Poland Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw Technical University, Wybrze_ze Wyspiańskiego 27, 50-370 Wroclaw, Poland Received 11 July 2005; accepted for publication 29 September 2005 Available online 2 November 2005 Abstract Fourier-transform Raman (FT-RS), infrared (FT-IR), and surface-enhanced Raman scattering (SERS) spectra of L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and L-Phe-L-AlaCH(OH)-PO3H2 (MD4) phosphonodipeptides adsorbed on colloidal silver surface have been measured. The respective vibrational band assignments have been proposed. The analysis of the SERS spectra shows that these peptides interact with the silver surface mainly through the aromatic ring of the Phe residue. In the case of MD1 and MD2 the aromatic ring seems to be almost parallel to the metal surface, while for MD3 and MD4 it links to the metal in perpendicular orientation. The analysis of the relative band intensities suggests a contribution from the CE (chemical-enhancement) mechanism to the MD2 SERS spectra. Additionally, characteristic vibrations of the phosphonate, amino, and methane groups to the SERS spectra indicate that these groups are involved in interaction with the silver surface. We suggest that the O–P–O fragment of MD3 and MD4 and the P@O fragment of MD1 and MD2 interact with the silver surface. Ó 2005 Elsevier B.V. All rights reserved. Keywords: Surface-enhanced Raman scattering, SERS; Fourier-transform Raman scattering, FT-RS; Fourier-transform infrared spectroscopy, FT-IR; Phosphonodipeptides * Corresponding author. E-mail address: [email protected] (E. Podstawka). 0039-6028/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.susc.2005.09.048 208 E. Podstawka et al. / Surface Science 599 (2005) 207–220 1. Introduction Vibrational spectroscopy using Fourier-transform Raman scattering (FT-RS) and infrared spectroscopy (FT-IR) is a universal and very sensitive method for investigating structural changes occurring in organic and biological compounds [1,2]. In the last two decades, a new Raman technique, the so-called surface-enhanced Raman spectroscopy (SERS)—due to its selectivity and sensitivity as well as a possibility to use a variety of metal substrates: i.e. Ag, Au, and Cu—has become a very powerful structural method for qualitative and quantitative analysis of: amino acids, peptides, water-soluble and membrane proteins, etc. [3–16] present in the solution at concentration of 103– 1010 M. What sets technique apart from other spectroscopic methods is that it is sensitive to the interactions of adsorbates with the metal colloid surface. That is why it provides insight into the orientation and distance of the functional groups, with respect to the metal surface, that are responsible for the adsorption process of the molecule. Moreover, if the orientation of adsorbed species changes their SERS spectra also changes. In this study, Fourier-transform Raman (FTRS) and infrared (FT-IR) methods are used to characterize the molecular structures of four phosphonodipeptides: L-Phe-L-Ala-PO3H2 (MD1), L- Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)PO3H2 (MD3), and L-Phe-L-Ala-CH(OH)-PO3H2 (MD4), where Phe denotes phenylalanine, Ala— alanine, and Val—valine. The under investigation peptides have been tested as the most potent inhibitors of cathepsin C [17], an enzyme that plays a key role in the activation of granule serine peptidases involved in inflammation, cell-mediated apoptosis, and tissue remodelling. Thus, it is believed that the inhibitors of this enzyme might be useful as possible drugs against rheumatoid arthritis and muscular dystrophy, as well as inhibitors of certain tumour metastases. Their molecular structures are shown in Fig. 1. The SERS study was performed in order to determine the nature of the interaction of these peptides with the silver surface, as well as to determine how the –CH(OH)-spacing unit and/or replacement of the L-Ala by the L-Val or b-Ala residue affect peptide physicochemical ability to adsorb on the silver surface. 2. Experimental 2.1. Peptide synthesis Phosphonodipeptides: L-Phe-L-Ala-PO3H2 (MD1) and L-Phe-L-Val-PO3H2 (MD2) were O H2N O PO3H2 N H2N N H H MD1 MD2 O H2N PO3H2 O PO3H2 N HO MD4 H H2N OH N PO3H2 H MD3 Fig. 1. Molecular structures of L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and L-Phe-L-Ala-CH(OH)-PO3H2 (MD4). E. Podstawka et al. / Surface Science 599 (2005) 207–220 synthesized according to the classical procedure [18], whereas L-Phe-b-Ala-CH(OH)-PO3H2 (MD3) and L-Phe-L-Ala-CH(OH)-PO3H2 (MD4) were obtained analogously to the previously described procedure elaborated for the synthesis of 2-amino-1-hydroxyalkylphosphonic acids [19] starting from appropriate N-protected peptidyl aldehydes. The purity and chemical structures of the peptides were proved by means of the 1H, 31 P, and 13C NMR spectra (Bruker Avance DRX 300 MHz spectrometer) and electrospray mass spectrometry (Finnigan Mat TSQ 700). 2.2. FT-Raman spectroscopy For FT-Raman measurements, about 1–2 mg of phosphodipeptide was placed in a glass capillary tube and measured directly (180° geometry). FTRS spectra were recorded on a Bio-Rad step-scan spectrometer (model FTS 6000) combined with a Bio-Rad Raman Accessory (model FTS 40) and liquid-nitrogen-cooled germanium detector. Typically, 1000 scans were collected with the resolution of 4 cm1. Excitation at 1064 nm was used from a Spectra-Physics continuum-wave Nd+3:YAG laser (model Topaz T10-106c). Power at the sample was maintained at 230 mW. No thermal degradation of the sample was observed during measurements. 2.3. FT-IR spectroscopy Thin pelettes containing 1 mg of each phosphonodipeptide dispersed in 200 mg of KBr were used for the FT-IR measurement. The spectra were recorded at room temperature as an average of 30 scans using a Brucker infrared spectrometer (model EQUINOX 55) equipped with a Nernst rod as the excitation source and a DT–GS detector in the 400–4000 cm1 range with the spectral resolution of 4 cm1. 2.4. SERS spectroscopy AgNO3 and NaBH4 were purchase from Sigma–Aldrich Co. (Poznań, Poland) and used without further purification. Solutions of the colloidal silver were prepared according to the standard procedure [14]. Briefly, 8.5 mg of AgNO3 209 dissolved in 50 mL deionized water at 4 °C was added drop-wise to the 150 mL of 1 mM solution of NaBH4, immersed in an ice-bath stirred vigorously. Once the addition of AgNO3 was completed, the resulting pale-yellow solution was stirred and maintained at 4 °C for approximately one hour. The excitation spectrum of the Ag sol prepared in this manner had an absorbance maximum at 396 nm. Sample solutions were prepared by dissolving the phosphodipeptide in deionized water. The concentrations of the samples before mixing with the colloid were set at 104 M. The final sample concentration in the silver colloid was 105 M. SERS spectra were obtained with a triple grating spectrometer (Jobin Yvon, T 64000). A liquid-nitrogen-cooled CCD detector (Jobin Yvon, model CCD3000) was used in these measurements. The spectral resolution was set at 4 cm1. The 514.5 nm line (30 mW at the sample) of an Arion laser (Spectra-Physics, model 2025) was used as the excitation source. No spectral changes due to the thermal degradation or desorption process were observed during these measurements. 3. Results and discussion 3.1. Fourier-transform Raman and infrared spectroscopy Fig. 2 presents the FT-RS spectra of MD1, MD2, MD3, and MD4 phosphonodipeptides in the frequency range of 140–1800 cm1. Fig. 3 shows the FT-IR spectra of the mentioned above compounds in the 400–1800 cm1 spectral range. So far, vibrational spectroscopy has not been used to study structures of phosphonodipeptides investigated here. A number of spectroscopic studies on L-phenylalanine [14,20,21], a- and b-alanine [22], valine, and their C-deuterated analogues [23], L-valine nitrate [24], tri-L-valine selenate [25], phosphate anions [26–29], L-phenylalanine L-phenylalaninium perchlorate and bis(DL-phenylalaninium) sulphate monohydrate [30], as well as L-phenylalanine-L-methionine and L-methionineL-phenylalanine [15] have been reported. Based on these results, and also on matrix-isolation 210 E. Podstawka et al. / Surface Science 599 (2005) 207–220 Fig. 2. FT-RS spectra of solid L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and 1 L-Phe-L-Ala-CH(OH)-PO3H2 (MD4) in the spectral range of 150–1800 cm . infrared and theoretical DFT and ab initio study of nonionized valine conformers [31], ROA investigation (Raman Optical Activity) of L-valine [32], and ab initio calculation of alanine [33], we have proposed band assignments for the FT-RS and FT-IR spectra of the investigated phosphonodipeptides included in Table 1. Below, we briefly discuss the characteristic Raman and IR bands crucial for the interpretation of SERS spectra, i.e. their clear assignment allows one to propose a way of interaction of the investigated molecules with the silver colloid. The FT-RS spectra (Fig. 2) of phosphonodipeptides investigated here show several characteristic bands that are clearly due to the aromatic ring vibrations of L-phenylalanine (Phe) [14,20,21,30]. These spectra are dominated by a band at 1004 cm1 that is accompanied by a band at around 1033 cm1. This very characteristic doublet is assigned to the ‘‘breathing’’ mode of the aromatic ring (the m12 mode) and the in-plane C–H bending (the m18a mode), respectively. Other bands associated with the Phe residue are observed at around 622, 1187, 1207, 1586, and 1606 cm1 (see Table 1 for detailed band positions). Their detailed assignments to the normal vibrations of the Phe ring are given in Table 1. In contradiction, the FT-IR spectra (Fig. 3) show only several the Phe ring modes discussed above. For example, the bands due to the m8a and m8b modes appear only in the FT-IR spectra of MD3 (1605 cm1) and MD4 (shoulder at 1586 cm1), respectively. Among the in-plane ring-bending modes, the m12 and m6b bands emerge in the FT-IR spectra. The former one is clearly seen around 1000 cm1 as a medium-intensity band for MD1 and MD4, as a weak shoulder for MD3, and is hidden under the 1041 cm1 band for MD2. The latter is observed at 629 cm1 only for MD4. In other compounds studied it is not seen due to its low absorption. E. Podstawka et al. / Surface Science 599 (2005) 207–220 211 Fig. 3. FT-IR spectra of solid L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and 1 L-Phe-L-Ala-CH(OH)-PO3H2 (MD4) in the spectral range of 400–1800 cm . In addition, the m9a and m15 modes with vibrational bands very close to each other (1186–1198 cm1) and a band of the m7a mode (1197–1216 cm1) overlap with the C–N symmetric stretching (ms(C–N)) and/or C–O deformation (d(C–O)) vibration (see Table 1). The medium-intensity bands observed in the range of 1297–1318 and 751–763 cm1 in the FTRS and FT-IR spectra may be assigned to other vibrations of the Phe residue, i.e. the sextant ring stretching (m14) and the out-of-plane C–H ring deformation (m11), respectively [34]. However, the assignment of the latter band is ambiguous because the C–C bending deformation mode (qb(C–C)), the C–P stretching (m(C–P)), and the –CH3 rocking vibration (qr(CH3)) are expected to be enhanced around this frequency. The m11 mode appears as a weak Raman band and a rather strong infrared band. This behaviour is associated with a large change in a dipole moment when the ring hydrogen atoms move in-phase out-of-plane of the Phe ring. Less characteristic features of the Phe ring are not going to be discussed in more detail. They are clearly listed in Table 1. The contribution of the aliphatic side-chain vibrations in the region between 1400 and 1800 cm1 has been thoroughly investigated. The medium-intensity bands near 1438–1450 and 1455–1464 cm1 in the discussed FT-RS (Fig. 2) and FT-IR spectra (Fig. 3) of MD1, MD2, and MD4 are readily assigned to the –CH2 group deformations (d(CH2)) and the –CH3 group antisymmetric bending deformation (das(CH3)) [2,35]. In the MD3 spectra, these two bands should be assigned to the deformations of the –CH2 group (d(CH2)) [22], since there is no methyl group in this compound. Two other medium-intensity bands around 1379–1393 and 1352–1372 cm1 are 212 E. Podstawka et al. / Surface Science 599 (2005) 207–220 Table 1 Frequency and proposed band assignments for FT-RS and FT-IR spectra of L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and L-Phe-L-Ala-CH(OH)-PO3H2 (MD4) Frequency [cm1] MD1 FT-RS 1661 MD3 IR FT-RS IR FT-RS IR FT-RS IR 1658 1659 1629 1676 1671 1661 1661 1606 1586 1605 1607 1584 1561 1586 das(NH2) Amide I Phe (m8a) Phe (m8b) 1538 Amide II 1498 1463 1438 1393 Phe (m19a) Phe (m19b) and/or das(CH3)/d(CH2) das(CH3)/d(CH2) Fermi resonance and/or d(CH)/qw(CH2) ds(CH3)/qt(CH2) d(CH)/qw(CH2) Phe (m14) Amide III and/or qw(CH2)) do.p.(CH), Phe (m3), and/or qt(CH2) qt(NH2) and/or qw(CH2) Phe (m7a), ms(C–N), and/or d(C–O) Phe (m9a), and/or d(CH2) ms(C–N) and/or m(P@O) Phe (m9b) and/or m(C–N) m(C–C) and/or qr(CH3) m(C–C), ms(C–N), and/or mas(PO3H2) Phe (m18b), m(C–PO3H2), and/or d(CH3) Phe (m18a) mas(PO3H2) Phe (m12) Phe (m5) and/or ms(PO3H2) qw(NH2) and/or qt(NH2) m(C–CH3) Phe (m10a), qr(CH2), and/or mas(O–P–O) m(C–C) and/or qb(NH2) Phe (m11), m(C–P), qr(NH2), and/or m(C–C) Amide V and/or qw(PO3H2)/q(CH2) m(C–C) and/or qr(H2O) Phe (m4), m(C–C), and/or d(C–H) qw(C–P–O) and/or d(PO3H2) Phe (m6b) qt(P–O) Phe (m9a), da(PO3H2), and/or qb(C–P–O) Phe (m9a) and/or qs(PO3H2) d(C–P–O) and/or qt(NH2) ds(PO3H2) d(C–C–N) and/or d(C–C–C) 1607 1586 1555 1558 1493 1497 1455 1450 1394 1446 1356 1301 1283 1256 1355 1301 1361 1300 1379 1361 1297 1256 1262 1258 1207 1187 1156 1213 1186 1169 1133 1209 1187 1157 1215 1080 1080 1048 1036 1004 982 917 MD4 1660 1633 1606 1586 1461 1450 Assignment MD2 1442 1154 1004 1146 1033 1102 1080 1051 1032 822 922 888 823 927 901 826 760 751 762 742 1197 1004 978 917 1464 1440 1393 1354 1318 1283 1352 1237 1220 1196 1164 1121 1094 1235 1216 1198 1178 1150 1119 1103 1078 1064 1282 1264 1208 1186 1159 1041 1033 1019 1000 1356 1318 1295 1268 1557 1540 1497 1455 1441 1404 1372 915 826 1036 1005 972 926 891 819 999 958 920 893 817 763 752 689 703 622 629 590 549 806 724 646 622 556 490 416 322 211 184 152 725 703 643 589 557 524 489 413 761 742 747 698 700 655 622 531 481 293 243 189 622 589 560 522 493 482 355 181 596 568 503 478 553 506 483 418 372 205 184 152 419 E. Podstawka et al. / Surface Science 599 (2005) 207–220 present in the FT-IR spectra and only one around 1350–1361 cm1 in the FT-RS spectra. The 1356 cm1 band is probably due to the C–H bending deformation of the phosphonodipeptide chain (d(CH)) for MD1, MD2, and MD4, while for MD3 this band may be associated with the –CH2 wagging vibration (qw(CH2)). With no doubt, the 1380 cm1 band is related to the das(CH3) mode. Moreover, the ligands FT-RS and FT-IR spectra (Figs. 2 and 3) investigated here exhibit a few bands that are due to the –NH2 group oscillations. Two of them, which appear in the range of 915– 927 and 478–493 cm1, are due to the –NH2 wagging (qw(NH2)) and twisting (qt(NH2)) vibrations, respectively [14,21,23,24,30,33]. The FT-IR spectra depict additional bands between 1658 and 1676 cm1 associated with the –NH2 antisymmetric deformation (das(NH2)) [14,21,23,24,30,33]. A band in the discussed spectra range of 742– 751 cm1 is assigned to the –PO3H2 wagging vibration (qw(PO3H2)) rather than to the amide V mode. Another, feature observed near 650 cm1 is due to the deformation of this group (d(PO3H2)). In addition, the 1080, 822, 549–568, 503–524, and 420 cm1 bands are assigned to the mas(PO3H2), mas(O–P–O), das(PO3H2)+db(C–P–O), qs(PO3H2), and ds(PO3H2) modes, respectively. Two other bands that may have a contribution from the phosphonate group vibrations are observed between 958–982 (ms(PO3H2)) and 1050 cm1 (m(C–PO3H2)) [22,26]. 3.2. Surface-enhanced Raman scattering Fig. 4 presents surface-enhanced Raman spectra (SERS) in the 350–1800 cm1 spectral range of all studied phosphonodipeptides adsorbed on the colloidal silver surface. The proposed SERS mode assignments given in Table 2 are based on the published data of amino acids [5,9,14,15], peptides [8,10,11], and phosphate anions [26] adsorbed on the colloidal or electrochemically prepared silver surfaces. The concentration of phosphonodipeptides in the Ag sol was 105 M. According to the TEM measurements, the average diameter of silver particles was 5–10 nm, so the concentration of phos- 213 phonodipeptides required for molecular coverage was estimated to be 106 M, assuming that the adsorbate was oriented perpendicularly to the colloidal silver surface [36]. This implies that the SERS spectrum corresponds to concentrations far above the full coverage limit. It should be mentioned that the SERS patterns of each of the four phosphonodipeptides investigated are different, although MD3 and MD4 do show some similarities. The SERS spectrum of MD4 seems to be the simplest one. Most of the strong bands are found in the 1000 and 1650 cm1 region, which points out a dominance of the in-plane bending and stretching vibrations of the phenyl ring in this SERS spectrum. The bands observed at 1002, 1030, 1205, 1583, and 1602 cm1 are due to the m12, m18a, m7a, m8b, and m8a modes of Phe, respectively, are downshifted only by a few wavenumbers in comparison to the corresponding FT-RS spectrum (Fig. 3) and show a higher full-width at the high maximum (FWHM). This red-shift and broadening of these bands suggest direct interaction of the Phe ring with the silver surface. In addition, the ratios of their SERS intensities are practically unaltered with regard to those observed in the Raman spectrum. It is noteworthy that the other Phe ring modes, i.e., m6b, m9b, m9a, and m19b, appear at 620, 1158, 1181, 1454 cm1 in the MD4 SERS spectrum as relatively weak bands. The intensity of the SERS bands depends of the square of the electric field. If z is taken as the direction perpendicular to the surface, and x and y are parallel to it, then this leads to a preferential enhancement by a factor of 4 in modes which contain a tensor component perpendicular to the surface relative to normal coordinates which do not contain a z component if the molecule is oriented perpendicularly to the surface. A maximum relative enhancement of 16:1 is expected for total symmetric modes which contain only a zz component relative to the modes containing a xy polarizibility vector only. Cerighton has shown that for aromatic amino acids with C2v symmetry, a flat adsorbed orientation is predicted via the selection rules to yield markedly different surface enhancement factors for these different symmetry mode types [36–38]. 214 E. Podstawka et al. / Surface Science 599 (2005) 207–220 Fig. 4. SERS spectra of L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and L-Phe-LAla-CH(OH)-PO3H2 (MD4) adsorbed at silver colloidal surface. Measurement conditions: sample concentrations in silver colloid, 105 M; excitation wavelength, 514.5 nm; power at sample, 30 mW. Specifically, the surface-enhanced factors for the A2, B1, and B2 vibrations are predicted to be approximately in the ratio of 4:1:1 for flat adsorption, and 1:4:4 for edge-on (on vertical) adsorption [36–38]. This large change in the enhancement factors for the A2 and B2 ring vibrations can be understood qualitatively from the ‘‘out-of-plane’’ and Ôin-plane’’ nature of these two symmetry types, respectively. For a lying ring orientation, therefore, the A2 vibrations contain a large polarizability tensor component normal to the surface than that for B2 vibrations, whereas the opposite should be the case for the vertical adsorption geometry. On the other hand, it is accepted that the SERS enhancement of the specific Phe ring modes is due to the formation of the p-complex of the aromatic ring with the metal surface [3,4,6,9,14]. If the Phe ring (C2v) is perpendicular to the silver surface modes of the A1, B1, B2 symmetry modes are preferentially enhanced (see Table 3 for Phe modes symmetry). However, in the case of the Phe ring lying horizontally on the surface, the A2 modes, together with the A1 and B1 modes, are preferentially enhanced [7,8]. For a flat-ring orientation, therefore, the A2 vibrations contain a polarizability tensor component normal to the surface larger than that for the B2 vibration, whereas the opposite should be the case for the vertical adsorption geometry. In general, A2 modes are very week in aromatic systems. This is due to the relatively small polarization changes which occur when the ring bends out of its plane. We are able to observe a B1 mode. The B1 modes contain the a xz polarizibility vector. The B2 modes are composed of the a yz vector. This precludes accurate determinations of orienta- E. Podstawka et al. / Surface Science 599 (2005) 207–220 215 Table 2 Frequency and proposed band assignments for SERS spectra of L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-AlaCH(OH)-PO3H2 (MD3), and L-Phe-L-Ala-CH(OH)-PO3H2 (MD4) Frequency [cm1] in SERS spectra MD1 1638 1602 1582 MD2 Assignment MD3 MD4 1638 1602 1587 1647 1601 1583 Amide I and/or das(NH2) Phe (m8a) Phe (m8b) 1514 1494 Amide II das(CH2) Phe (m19a) 1536 1503 1607 1592 1569 1539 1513 1452 1479 1462 1443 1404 1393 1347 1270 1235 1206 1171 1362 1454 1438 1402 1382 1364 1206 1182 1162 1205 1181 1158 1101 1122 1030 1002 961 911 872 850 829 1030 1002 761 703 619 572 761 702 620 489 408 485 413 1534 1378 1253 1208 1160 1142 1084 1044 1033 1002 955 912 857 828 748 720 620 561 493 1136 1100 1080 1046 1031 1003 956 916 856 824 803 761 702 621 565 527 492 404 tions at the surface. The A1 modes are very strong in the Raman spectra of aromatic compounds. The A1 modes are difficult to analyze for their orientation since they are composed of linear combinations of xx, zy, and zz. Simple geometrical arguments show that if the molecule is oriented with the ring perpendicular to the surface, then the intensity ratio of A1/B1 must be 1.155 [39], where of A1 indicates m12. 913 876 852 829 Phe (m19b) and/or ds(CH2)/d(CH2) das(CH2)/d(CH2) Fermi resonance, and/or das(CH3)/qw(CH2) di.p.(CH2–CH2)/qt(CH2) ds(CH3)/qw(CH2) m(P@O), and d(C–Ca–H) Phe (m3) Phe (m7a) Phe (m9a) and/or d(CH2) Phe (m9b) and/or ms(C–N) m(P@O) qr(CH3) m(C–NH2) and/or mas ðPO2 3 Þ Phe (m18b), m(Ca–N) and/or m(C–P) Phe (m18a) Phe (m12) Phe (m5), ms ðPO2 3 Þ, and/or m(C–CH3) mðPO2 3 Þ m(C–C) ms(C–N–C) and/or m(C–C) mas(O–P–O) and/or Phe (m10a) m(C–CH3)isopropoyl Phe (m1), Phe (m11), and/or ms(O–P–O) d(NH2–C–(O@)C–NH) Phe (m6b) Phe (m1), das ðPO2 3 Þ and/or qb(C–P–O) qt(NH2), and/or d(C–C–N) Phe (m16a) and/or d(C–C–N) The SERS spectrum usually shows a large enhancement of the 405–420 cm1 mode. This is to be expected since this mode is mostly composed of the a zz tensor [39]. A comparison of the SERS spectra of the surface product with the silver surface shows several trends. The intensity ratio of the 1000 cm1 band relative to the 470 cm1 band is about one. This is predicted for molecules which are perpendicular to the surface. 216 E. Podstawka et al. / Surface Science 599 (2005) 207–220 Table 3 Frequency and allocation to normal coordinates of phenyl ring vibrations SERS Assignments A1 3060 1591 1483 1199 1175 1024 998 787 1205 2 8a 19a 13 9a 18a 12 1 7a axx ayy azz A2 848 401 978 10a 16a 17a axy Out-of-plane Out-of-plane Out-of-plane B1 383 547 690 775 925 987 15 16b 4 11 17b 5 axz i azx Out-of-plane Out-of-plane Out-of-plane B2 626 383 1289 1337 1448 1584 506 6b 3 4 14 19b 8b 10b ayy In-plane In-plane In-plane In-plane In-plane In-plane In-plane In-plane In-plane Out-of-plane ayz i ayx ayz In-plane In-plane axz Out-of-plane According to these findings, in the SERS spectrum of MD4, the enhancement of the 1002 cm1 band of the m12 mode, together with the m6b, m9b, m19b, and m8b modes of the B2 symmetry at 620, 1158, 1454, and 1583 cm1, respectively, suggests that in this case the Phe ring adopts almost a vertical position in respect to the silver surface. Also, a lack of a significant frequency shift of m12 suggests that the aromatic ring does not lie flat on the silver surface [39]. The bands attributable to the L-Ala residue as well as to the amino and phosphonate groups can be also traced in the MD4 SERS spectrum presented here. The –CH3 and –CH2 group deformation modes appear as spectral features at 1122, 1364, 1382, 1402, 1454, and 1514 cm1. All these bands are relatively weak, except the one at 1454 cm1, which exhibits medium-intensity. These bands are due to the –CH3 group rocking (qr(CH3)), the –CH3 symmetric deformation (ds(CH3)), the CH2–CH2 in-plane deformation (di.p.(CH2–CH2)), the –CH3 antisymmetric deformation (das(CH3)), and the –CH2 symmetric (ds(CH2)) and antisymmetric (das(CH2)) deformation vibrations, respectively. The appearance of these bands, as well as the analysis of their intensities suggest that the methylene group adjacent to the Phe ring is in close proximity to the silver surface rather than interacting with it directly as shown in Fig. 5. In addition, the occurrence of the bands of the C–NH2 and C–N–C moieties at 413, 485, 702, and 852 cm1 assigned to the d(C–C–N), qt(NH2)+d(C–C–N), d(NH2– C–(O=)C–NH), and ms(C–N–C) modes, respectively, indicates that the C–NH2 group accepts a position near the silver surface rather than interacting with it, since the above-mentioned bands are only weakly enhanced in the MD4 SERS spectrum. In addition, the relatively weak bands observed at 761, 829, and 913 cm1 due to the O–P–O fragment symmetric and antisymmetric stretching, and the –PO2 3 symmetric stretching, respectively, also appear in the SERS spectrum of MD4. It was shown that the phosphate and phosphonic anions can adopt several possible geometries on electrode surfaces [26]. In the pH conditions of our experiment, it is the most probable that the tridentate ðPO2 3 Þ anion interacts with the surface, giving rise to the MD4 SERS features at the above-mentioned frequencies. In general, the presence of the 1250– 1270, 1140, 914, and 565 cm1 bands in the SERS spectrum of the phosphate residue containing molecules points out that the P@O fragment of the phosphate group interacts with, or is in close proximity to, the surface. However, the presence of the 914, 828, 761, and 565 cm1 bands suggest that the O–P–O fragment participates in the molecule interaction with the surface. Thus, in the SERS spectrum of MD4 presented here, the most enhanced are the bands due to the O– P–O vibrations and no those due to the P@O vibrations. We conclude that MD4 interacts with E. Podstawka et al. / Surface Science 599 (2005) 207–220 217 Fig. 5. Manner of interactions of L-Phe-L-Ala-PO3H2 (MD1), L-Phe-L-Val-PO3H2 (MD2), L-Phe-b-Ala-CH(OH)-PO3H2 (MD3), and L-Phe-L-Ala-CH(OH)-PO3H2 (MD4) with the silver surface. the silver through the –PO2 2 fragment, while the P@O fragment is rather remote from the surface. The last band that should be assigned for MD4 is the amide I band. A spectral feature observed at 1647 cm1 is due to this vibration. Summarizing, the pattern of the SERS spectrum of MD4 points out that the Phe ring of this phosphonodipeptide adopts a parallel orientation with regard to the silver surface, and the O–P–O fragment is directly involved in the interaction with the silver surface. In addition, the MD4 backbone lies on the surface in such a way that the –NH2 and –CH3 side-chain groups occur near the surface. This manner of MD4 binding to the silver surface is presented in Fig. 5. The pattern of the SERS spectrum of MD3 resembles that of MD4 (see Fig. 4). Therefore, we gather that this posphonodipeptide interacts with the colloidal silver surface in similar a manner as MD4. This means, that the replacement of the L-Ala residue by b-Ala residue does not change the overall mechanism of the interactions of these phosphonodipeptides with the colloidal silver surface. Upon analysis, the m12, m18a, m7a, and m8a modes of the Phe ring appear in the MD3 SERS spectrum almost at the same frequency as those in the FT-RS spectrum (see Table 2 for detailed frequencies), and are one of the most intense bands of the discussed spectrum, again as in the FT-RS spectrum. In addition, the m6b, m9b, and m8b modes of the B2 symmetry are enhanced (Table 2). This constituents evidence that the Phe ring adopts a perpendicular orientation with regard to the silver surface. On the other hand, the bands observed at 408, 489, 703, 850, and 1101 cm1 in the discussed SERS spectrum are due to the different vibration of the C–NH2 and C–N–C moieties (Table 2). Both, the appearance of these bands in the MD3 SERS spectrum and their weak intensities, indicate that the MD3 backbone is spread over the surface in such a way that the –NH2 group and the C–N– C unit are in lose contact with the silver surface or in close proximity to it. A confirmation of these findings is providing by the bands at 1443, 1393, and 1362 cm1 due to the deformation vibrations of the –CH2 and CH2–CH2 moieties, as well as the 872 cm1 band of the m(C–C) mode. Furthermore, band observed at 572, 761, 829, and 218 E. Podstawka et al. / Surface Science 599 (2005) 207–220 911 cm1 in the discussed MD3 SERS spectrum are assigned to the different phosphonate group vibrations. Two of these bands, at 761 and 829 cm1, are due to ms(O–P–O) and mas(O–P–O), respectively. Their presence suggests that the O–P–O fragment is involved in the interaction with the silver. On the other hand, the other two bands, namely at 572 and 911 cm1 due to 2 das(PO2 3 )+qb(C–P–O) and m(PO3 ), respectively, point to a weak participation of the P@O fragment of the phosphonate group in this interaction. However, the fact that no band due to m(P@O) is present may suggest that the P@O unit is rather remote from the surface. The amide I bands appear for MD3 (1638 cm1) at a similar position as in the SERS spectrum of MD4. In addition to this band, the amide II band is enhanced at 1534 cm1. To recapitulate, the manner of MD3 binding to the silver surface is presented in Fig. 5. A slightly different mechanism of interaction with the silver surface is observed for MD1 and MD2, which is evident from them SERS spectra. In the MD2 SERS spectrum, just as in the MD3 and MD4 SERS spectra, bands due to the Phe ring modes are enhanced, i.e. the bands 621 cm1, m6b; 1003 cm1, m12; 1031 cm1, m18a; 1171 cm1, m9a; 1206 cm1, m7a; 1235 cm1, m3; 1592 cm1, m8b; and 1607 cm1, m8a. However, these bands, except m8b, exhibit low intensity, especially that of the m12 mode, compared to the corresponding FTRS spectrum, which may indicate that the Phe ring is tilted or lying approximately flat on the silver surface. In addition, only two of the above-mentioned modes have the B2 symmetry, namely m6b and m8b, supporting the above conclusions. The former is very weak in the MD2 SERS spectrum, while the latter is the most intense band of the spectrum. The SERS excitation profiles show that enhancement of the Phe aromatic ring vibrations may be due to both electromagnetic (EME) and chemical mechanisms (CE) [11]. On the basis of the selection rules of the EME mechanism, the totally symmetric ring modes should be most enhanced when the Phe ring is almost perpendicular to the metal surface. On the other hand, a contribution of the CE mechanism in the enhancement mechanism is manifested by the strong enhancement of the band of the m8 mode. In the SERS spectrum of MD2, the 1592 cm1 band is the strongest band in the spectrum. Therefore, it can be concluded that the CE mechanism plays an essential role in the case of MD2. The bands attributable to the L-Val residue and to the amino and phosphonate groups can be also traced in the presented SERS spectrum of MD2. The isopropyl group of the side-chain of L-Val gives rise to the several bands observed at 803, 956, 1100 cm1, and in the 1347–1513 cm1 range. A detailed assignment of these bands is given in Table 2. It is worth mentioning that two of these bands observed at 1347 and 1404 cm1 and due to the ds(CH3) and das(CH3) modes, respectively, are relatively strongly enhanced. This is probably because the isopropyl group points at the surface and interacts with it. In addition to these bands, the 404, 492, 702, 1046, and 1080 cm1 spectral features due to the different vibrations of the C–NH2 and C–N–C moieties appear in the SERS spectrum of MD2. The last two above-mentioned bands of the Ca–N and Ca–NH2 stretching vibrations, respectively, do not appear in the SERS spectra of MD3 and MD4 previously discussed, and are of low intensity in the MD2 SERS spectrum. This observation indicates that the C–NH2 and C–N–C moieties of MD2 participate in the weak interaction of this phosphonodipeptide with the silver surface, while these moieties in MD3 and MD4 are in close proximity to the silver surface. The P-terminal group of MD2 also contributes to the MD2 SERS spectrum. As in the case of MD3 and MD4, in the MD2 SERS spectrum the different PO32 and O–P–O deformation vibrations are enhanced in the frequency range of 565–916 cm1 (see Table 2), pointing out that the O–P–O group is in close proximity to the silver surface. In addition to these, the bands due to the P@O stretching vibrations appear at 1136 and 1270 cm1, respectively. The presence of these two bands suggests that the P@O fragment binds to the silver. Surprisingly, no amide I band is enhanced in the MD2 SERS spectrum. This is probably because the amide bond lies flat on the silver surface. However, the spectral feature at 1539 cm1 is due to the amide II mode. E. Podstawka et al. / Surface Science 599 (2005) 207–220 Summarizing, MD2 interacts with the silver surface mainly through the Phe ring that seems lie almost flat on the surface, and the P@O fragment of the other side of the phosphonodipeptide backbone. These interactions probably force the amide bond to lie flat on the surface. Additionally, in this conformation of MD2 its amino and isopropyl groups of the side-chain interact with the surface. The stale of MD2 binding to the silver surface described above is shown in Fig. 5. The last phosphonodipeptide, MD1, investigated here behaves in another way. Again, the bands due to the Phe ring modes in the SERS spectrum of MD1 are relatively weakly enhanced, i.e., m6b, m12, m18a, m9a, m7a, m8b, and m8a (Fig. 4). This phenomenon, together with the low intensity of the 620 (m6b) and 1582 cm1 (m8b) bands of B2 symmetry, indicates that the phenyl ring adopt a tilted orientation with regard to the silver surface. The roughly similar orientation of the Phe ring on the silver has been already shown for MD2, although the CE mechanism plays an essential role in MD2 enhancement as was discussed above. In the case of MD1, we do not observe any bands intensification for the m8 mode. As a matter of fact, the m8 modes are weakly enhanced. This is why we assume that the EME mechanism is responsible for the enhancement of the Phe ring modes in MD1. This difference in enhancement between MD1 and MD2 is probably a result of a different tilt/angle of the Phe ring regarding to the silver surface. On the other hand, the relatively strong band at 720 cm1due to the NH2–C–(O@)C–NH deformation vibrations and two bands of medium intensity at 1044 and 1084 cm1 assigned to m(Ca–N) and m(Ca–NH2) provide the evidence for the direct interaction of the NH2–C–(O@)C–NH fragment with the silver. In addition, some weak bands allocated to the –CH3 vibrations observed in the 1378– 1503 cm1 range and a band at 955 cm1 may suggest that this group is in close proximity to the surface (see Table 2 for detailed band frequencies and their assignment). It should be mentioned that the most intense band of the MD1 SERS spectrum at 1253 cm1 is due to the m(P@O) mode. This tells us that the P@O fragment strongly binds to the silver. This 219 is supported by the other m(P@O) band at 1142 cm1 and a band at 561 cm1 assigned to das(PO2 3 )+qb(C–P–O). Furthermore, it seems that the O–P–O fragment is remote from the surface because bands at 748 and 828 cm1 allocated to its symmetric and antisymmetric deformations, respectively, show relatively weak intensities. Other observed bands can be assigned to the amide and backbone modes. Spectral features at 1536 and 1638 cm1 are assigned to the amide II and I, respectively. Summarizing, Fig. 5 presents a way of MD1 binding onto the silver surface. 4. Conclusion Phosphonodipeptides investigated in this work adsorb on the colloidal silver surface mainly via the aromatic ring of the Phe residue, which in the MD1 and MD2 case adopts a tilted or closeto-flat orientation with regard to the surface, while for MD3 and MD4, the Phe ring accepts a perpendicular orientation. The analysis of the relative band intensities of the m8 modes allows us to establish the contribution of the CE mechanism to the MD2 SERS spectra. In addition, we find that the phosphonate (–PO2 3 ), amino, and methane groups are involved in the adsorption process of these phosphonodipeptides on the silver surface. This phenomenon is clearly seen for the PO2 group, 3 which in the case of MD3 and MD4 interacts with the silver through its O–P–O fragment, while the P@O unit is rather remote from the surface. The opposite situation is observed for MD1, where the P@O fragment strongly binds to the surface. Mean while, for MD2 the PO2 group interacts 3 mainly via P@O as well as O–P–O. Moreover, the backbone of phosponodipeptides all investigated here lies on the silver surface in such a way that the NH2–C–(O@)C–NH fragment of MD1, the isopropyl and amine groups of MD2, the –CH2–CH2 and CNH2 fragments of MD3, and the methane group (the amine group seems to be in close proximity to the surface) of MD4 are involved in the interaction with the silver. 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