Surface-enhanced Raman spectroscopy of tridehydropeptides adsorbed on silver electrode

Journal of Molecular Structure 1065-1066 (2014) 143–149

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Surface-enhanced Raman spectroscopy of tridehydropeptides adsorbed on silver electrode Mariusz Gackowski a,b, Kamilla Malek a,⇑ a b

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Peptides containing dehydroalanine and

two isomers of dehydrophenylalanine are studied.  The structure of the peptides is investigated by using IR, Raman and SERS techniques.  The adsorption mechanism of the peptides on the silver electrode is proposed.

a r t i c l e

i n f o

Article history: Received 14 January 2014 Received in revised form 25 February 2014 Accepted 25 February 2014 Available online 11 March 2014 Keywords: Dehydroalanine Dehydrophenylalanine Tripeptides SERS Ag electrode

a b s t r a c t Surface-enhanced Raman spectroscopy (SERS) was used to characterise interactions between six tridehydropeptides and the silver electrode. Boc-Gly-X-Gly-OMe and Boc-Gly-X-Gly-COOH (X = dehydroalanine (DAla), dehydrophenylalanine (D(Z)Phe and D(E)Phe) were studied in this work. The type of the rigid dehydroamino acid residue and the isomer of DPhe have a strong impact on the adsorption mechanism of the peptides. The respective vibrational assignments were proposed by the analysis of FTIR and FT-Raman spectra of solids enabling the evaluation of SERS spectra. Generally, the most intensive SERS bands relative to those in the bulk-phase spectra are associated with vibrations of the dehydroamino acid moiety, i.e. the C@C bond and the phenyl ring. Only, in the case of the peptides containing the DAla and D(Z)Phe residues and ionised carboxylate group, the molecules interact with the silver electrode via the peptide backbone. In most cases of the peptide containing DPhe the aromatic ring is almost perpendicular to the metal surface. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Dehydroamino acids are formed by changing sp3 hybridization of the Ca carbon. The formed double CaCb bond promotes the steric hindrance and imposes conformational restrictions in peptides. In addition, this leads to p-electron conjugation between the CaCb ⇑ Corresponding author. Tel.: +48 12 663 2064; fax: +48 12 634 0515. E-mail address: [email protected] (K. Malek). http://dx.doi.org/10.1016/j.molstruc.2014.02.050 0022-2860/Ó 2014 Elsevier B.V. All rights reserved.

bond and amide groups. This modified amino acid residues have been found in a number of naturally occurring peptides like in nisins [1] or thiopeptide antibiotics [2] and their action is also connected with biocatalysis [3,4]. The most common in nature dehydroamino acids are dehydroalanine (DAla) and (Z)dehydrophenylalanine (D(Z)Phe). The E form of DPhe shows the lower thermodynamic stability than the Z analogue and is less common in natural peptides. It is expected that both the isomers possess different conformational structures, and therefore they often serve

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as pharmacophores [5]. However, details on mechanism of their pharmacological action have not been yet recognised. Fourier transform infrared absorption (FTIR) and Raman scattering (RS) spectroscopies are well known as a powerful tool in studies on the secondary structure of peptides and proteins in the solid state and solutions by investigating the spectral amide I, II and III regions sensitive to the peptide backbone structure. Several experimental [6–11] and theoretical [6,8,11] studies on molecular structure of peptides containing DAla and D(E/Z)Phe have been reported. But they have been mainly focused on the analysis of IR [6–9], circular dichroism [7,10] and NMR spectra [11]. The SERS spectra of various kinds of peptides have been also reported up to now [12–16]. However, to our best knowledge, a tentative Raman and SERS study of dehydropeptides of various number of dehydroresidues and terminal groups has not been reported except of our recent study [17]. In this work we reported IR, Raman and SERS spectra of three dipeptides Boc-Gly-X, where X is DAla, D(Z)Phe and D(E)Phe. SERS spectra were collected by using Ag sol as a metal substrate. In this case, the dehydropeptide–metal interactions mainly occur due to the deprotonation of the terminal carboxylic group. The adsorption process strongly affects the appearance of SERS bands in the region of 1500– 1650 cm1, indicating possibility of p-electron resonance between the phenyl ring and the peptide backbone. SERS technique was also used in studies on pentapeptides containing DPhe [18]. This investigation showed that this type of peptides is promising a capping agent for gold nanoparticles exhibiting properties of drug delivery vehicles. In the present study, we focus on an evaluation of SERS features of tridehydropeptides, in which a rigid central moiety (DAla, DPhe) is surrounded by two flexible glycine residues. The following peptides are chosen as model compounds: Boc-Gly-DAla-Gly-OMe (P1), Boc-Gly-D(Z)Phe-Gly-OMe (P2), and Boc-Gly-D(E)Phe-GlyOMe (P3) (Boc, t-butoxycarbonyl; OMe, methoxy), see Scheme 1. In addition, we investigate SERS profile of their structural analogous, in which the OMe group is substituted by the COOH group (P10 , P20 , and P30 , respectively) since it is well known that this group exhibits the high affinity to the metal surface [17,23].

Despite this the chosen molecules exhibit a variety of conformational preferences in the solid state that may be reflected in their ability to adsorb on the metallic support. Thus, we also discuss FTIR and Raman profile of the solids to give an insight into molecular structures of the peptides. The main aim of this work is to determine the nature of the interaction of these peptides with the solid silver surface as well as to determine how the type of dehydroamino acid residue and the C-terminal functional group affect peptide ability to adsorb on the silver electrode. 2. Experimental Compounds were synthesized according to the procedure described in [19]. Briefly, peptides were synthesized in condensation reaction between trifluroacetate (TFA) amide of alanine or phenylalanine and a-keto acid (pyruvic acid or phenylpyruvic acid) in benzene. The reaction is catalysed by p-toluenesulfonic acid. In the case of TFA-Gly-DPhe-Gly, both isomers (Z and E) are formed in ratio of 4:1. Then, the TFA group is substituted by the Boc group. Yield for all syntheses: 50–80%. The purity of the compounds were tested by standard analytical methods (elemental analysis and NMR). For FT-Raman measurements, a few milligrams of each solid sample were measured on metal discs directly. Spectra were accumulated from 256 scans, with a spectral resolution of 4 cm1. Spectra were recorded on a MultiRAM FT-Raman Spectrometer (Bruker), equipped with a germanium detector cooled with liquid nitrogen. Each of the samples was illuminated from a Nd:YAG laser (k = 1064 nm) at an output power of 100 mW. ATR FTIR spectra of the solid samples were collected using a ALPHA Bruker spectrometer equipped with a 1-reflection ATR diamond crystal. Spectra were collected in the range of 375–4000 cm1, with spectral resolution of 4 cm1. 128 scans were co-added, and then extended ATR correction was employed. For SERS measurements, solids were dissolved in ethanol/water in volume ratio of 1:1 to prepare 1  103 M solutions. After dissolving pH of solutions was ca. 6. Since, protonation constant of the COOH group in dehydropeptides is found in the range of 3–4, this group should be deprotonated in P10 –P30 during collection of SERS spectra [20]. The silver electrodes were prepared by electrochemical roughening, using five positive/negative cycles in 0.1 M KCl solution from 0.3 to +0.3 V (vs Ag/AgCl) and the potential close to the end of the last negative cycle was held for 30 s. A platinum electrode was used as a counter electrode while Ag/AgCl in 1 M KCl was used as a reference. The silver electrodes were immersed in 1  103 M solutions of each peptide, then SERS spectra were recorded after a few hours since no SERS signal was observed immediately after immersing the electrode in the solution. From SEM pictures one can deduce that the electrode is not roughened equally on its whole surface (Fig. 1A and B). Roughness of the silver surface also varies in thickness. The SERS measurements were conducted on a LabRam 800 Raman Spectrometer equipped in a confocal microscope, a CCD detector and a He–Ne laser excitation line (632.8 nm). The spectra were collected through an air objective with the magnification of 50 with the use of 600 g/mm grating. For each SERS spectrum 60 scans were collected with integration time of 1 s. Output laser power was set from 0.154 to 3.4 mW. Laser power for each measurement of SERS spectrum was adjusted in such a way to use a minimal power and to obtain spectrum showing a good signal to noise ratio. 3. Results and discussion

Scheme 1. Structures of the tripeptides P1–P3.

Figs. 2–4 show FT-Raman spectra of the peptides P1–P3 and their analogues P10 –P30 in the solid state along with SERS spectra collected on silver electrodes.

M. Gackowski, K. Malek / Journal of Molecular Structure 1065-1066 (2014) 143–149

Fig. 1. SEM photographs of the roughened silver electrode used in SERS under 33 (A) and 16,000 (B) magnifications.

In the case of Boc-Gly-DAla-Gly-OMe (P1), SERS spectra showed different spectral profiles during measurements from various points of the electrode exhibiting likely photodegradation process of the peptide due to excitation by laser light (data not shown). It is worth-mentioning that we attempted to collect SERS spectra of P1 on silver colloids and laser excitations in the Vis and NIR region, but no SERS signal or large variability of signal were observed during measurements. The selected band’s positions in FT-Raman and SERS spectra with their tentative assignments are collected in Tables 1–3 [9,12–17,21,22]. Here, we assign only major peaks, which appear in SERS spectra, to give an insight into the adsorption mechanism of the studied dehydropetides. FTIR and FT-Raman spectra of P1–P3 are discussed in detail in [9], while ATR FTIR spectra of P10 –P30 are present in Fig. 5. The comparison of FT-Raman spectra of the peptides P1 and P10 , especially in the region of 1550–1740 cm1 (typical for the amide I and C@C vibrations), indicates significant differences in a secondary structure of both compounds (c.f. Fig. 2). A detailed examination of FTIR and FT-Raman spectra of P1 exhibited spectral markers specific for an extended conformation of the peptide confirmed by its crystallographic structure [9]. Amide I bands were observed at 1690 and 1660 cm1 in IR spectrum of P1 [9], whereas three IR bands at 1677, 1656, and 1632 cm1 are present in the ATR FTIR spectrum of P10 (Fig. 5). The latter indicates that due to the substitution of the OMe group in P1 by the COOH group in P10 , the tridehydropeptide P10 adopts a folded structure. This is also confirmed by the IR position of the amide II band at 1504 cm1 accompanied by a few shoulders at 1560 and 1537 cm1 (c.f. Fig. 5). The appearance of a large number of amide II bands can be associated with variation in dihedral angles in the peptide

145

Fig. 2. FT-Raman spectra of Boc-Gly-DAla-Gly-OMe (P1) and Boc-Gly-DAla-GlyCOOH (P10 ) in the solid state (black trace) and SERS spectrum of P10 on the Ag electrode (red trace) (laser excitation: 632.8 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

backbone. In turn, SERS spectra of both peptides suggest an impact of this substitution on interactions between the metal and the dehydropeptides containing dehydroalanine. As mentioned above, P1 adsorbed on the silver electrode is probably decomposed during exposure to laser light, whereas a good quality SERS spectrum was recorded for P10 (see Fig. 2). Vibrations assigned to SERS bands of P10 are collected in Table 1. The principal bands in the SERS spectrum of P10 are those corresponding to the amide II and amide III vibrations, the symmetric stretches of the deprotonated carboxylic group, and the bending modes of the glycine residues. The amide II and amide III are quite distinctive, signifying that the amide groups of the peptide are not perpendicular to the silver surface since both are the combination of the stretching and bending vibrations. The presence of these bands were also identified in SERS spectrum of the P10 analogue with a shorter backbone, i.e. Boc-Gly-DAla-COOH [17]. However the amide I mode was more intensified than other amide vibrations in SERS of this dehydropeptide on the contrary to P10 . This can indicate that the amide moieties are tilted with respect to the metal electrode. The observed shift of the SERS amide bands from their counterparts in the FT-Raman spectrum of the solid sample (15–20 cm1) may in addition suggest some changes in the secondary structure of P10 . According to the previous studies [6,9,17], the high-wavenumber amide II bands are specific for a a-helical structure typical for the Boc-Gly-moiety, while a band at 1508 cm1 appears due to a turn conformation around the dehydroamino acid residue (DAla). The bands at 1581 and 1547 cm1 exhibit a higher SERS intensity than the 1508 cm1

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Fig. 3. FT-Raman spectra of Boc-Gly-D(Z)Phe-Gly-OMe (P2) and Boc-Gly-D(Z)PheGly-COOH (P20 ) in the solid state (black trace) and their SERS spectra on the Ag electrode (red trace) (laser excitation: 632.8 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. FT-Raman spectra of Boc-Gly-D(E)Phe-Gly-OMe (P3) and Boc-Gly-D(E)PheGly-COOH (P30 ) in the solid state (black trace) and their SERS spectra on the Ag electrode (red trace) (laser excitation: 632.8 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

band (Fig. 2). We interpret the relative intensity of these bands as an evidence that the modes of a a-helical conformation have components of Raman polarizability perpendicular to the metal surface. Since, the largest shift is found for the bands assigned to the helical structure, likely the conformation of the Boc-Gly-fragment of P10 changes due to the adsorption on the silver. This is also confirmed by the 9 cm1 red-shift of the amide III band at 1252 cm1 attributed to helical structures [9]. The other feature of the SERS spectrum of Boc-Gly-DAla-COOH is an enhanced band of the >C@CH2 stretching mode at 1621 cm1 [17], however, this band is not present in the SERS spectrum of Boc-Gly-DAla-GlyCOOH. For the latter, we only observe the intensification of the scissoring mode of the @CH2 group at 1421 cm1, suggesting that blocking the dehydro moiety by the second Gly residue prevents from a perpendicular orientation of the DAla group on the electrode. In addition, the deprotonated C-terminal group often participates in the adsorption on the metal through its delocalized p-electron system [17,23]. This is verified by the presence of a SERS band in the region of 1360–1420 cm1 that is attributed to the symmetric stretching vibration of the COO group. In the SERS spectrum of P10 , we assign a SERS band at 1379 cm1 of a medium intensity to this mode. Since, the protonated molecules of the dehydropeptide are present in solution (pH 6) used in SERS measurements, the appearance of this band indicates the removal of the carboxylic proton due to the interaction with the metal. According to Fleger and co-workers [23], the orientation of the carboxylic group can be identified by relative intensity of SERS bands assigned to the stretching and bending vibrations of the COO group. For P10 , both bands at 1379 and 676 cm1, respectively,

Table 1 Positions of ATR FTIR, FT-Raman, and SERS bands (in cm1) of Boc-Gly-DAla-GlyCOOH (P10 ) together with the proposed assignment [9,12–17,21,22]. ATR FTIR

FT Raman

1677m 1656vs 1632m 1560w,sh 1537m,sh 1504vs

1678vs 1658m,sh 1628m 1559vw 1532vw 1510w 1454m 1411m 1375vw 1322w 1263s 1247m,sh 1093m 1059s 681vw

1418s 1370s 1260m

1062m 678m

SERS

Modea

1581s 1547s 1508s 1448m 1421s 1379m 1326vs 1252m 1231m 1107m 1074m 676m

Amide I Amide I Amide I Amide II Amide II Amide II d(CH2)Gly d(@CH2) ms(COO), x(CH2)Gly Amide III Amide III s(CH2)Gly m(CN), mas(COC)Boc q(CH3)Boc, x(C-O)Boc x(COO)

a m, stretching; d, scissoring; x, wagging; s, twisting; q, rocking; as, asymmetric; vs very strong; s, strong; m, medium; w, weak; vw, very weak; sh, shoulder.

exhibit similar intensity, thus the carboxylate anion adopts a bent orientation preventing a direct interaction of its delocalised p-electron system with the metal surface. The introduction of the bulk dehydrophenylalanine between the glycine residues leads to completely different IR, Raman and SERS features in the comparison to the DAla moiety (see Figs. 2–5). The change in the type of the isomer from Z to E also affects molecular structure of a peptide, and consequently vibrational spectra. The

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Table 2 Positions of ATR FTIR, FT-Raman, and SERS bands (in cm1) of Boc-Gly-D(Z)Phe-Gly-OMe (P2) and Boc-Gly-D(Z)Phe-Gly-COOH (P20 ) together with the proposed assignment [9,12– 17,21,22]. P20

P2 ATR FTIR

FT-Raman

1685s 1656vs 1640m,sh 1620m

1688m 1662s 1640s 1623vs 1604vs

1598s 1565m,sh

1558m,sh

1457w

1453vw

ATR FTIR

FT-Raman

1664s 1650vs

1666m 1653s 1606vs 1599vs

1601vs 1523s 1493s

1528vw

1443m

1448w

1328m 1281m 1207s 1101w

1206w 1120w 1003m

1001s

838w 655s a

1596vs 1564m 1537m 1478s

1314vw 1290w 1206vs 1101vw 1003s 844vw

1445m 1362s 1353s

1200m 1102m 1000m 837w 654w

Modea Amide I Amide I Amide I m(C@C) 8a Amide II Amide II Amide II d(CH3)Boc d(CH2)Gly ms(COO) Amide III Amide III 13 m(CN), mas(COC)Boc 12 m(CC), x(COO) 5

m, stretching; d, scissoring; x, wagging; s, symmetric; as, asymmetric; vs very strong; s, strong; m, medium; w, weak; vw, very weak; sh, shoulder.

Table 3 Positions of ATR FTIR, FT-Raman, and SERS bands (in cm1) of Boc-Gly-D(E)Phe-GlyOMe (P3) and Boc-Gly-D(E)Phe-Gly-COOH (P30 ) together with the proposed assignment [9,12–17,21,22]. P30

P3 ATR FTIR

FT-Raman

SERS

1695vs 1655vs 1629s

1543s 1511vs 1301m 1268m 1252s

ATR FTIR

FT-Raman

SERS

1708 1686m 1654w 1633vs 1600vs

a

SERS

1468w

1321w 1255w 1211vs

1252m,sh 1210s

SERS

1654s 1636w 1602s

1638vs 1601s

1601s

1533vs 1304m 1268vw 1255vw 1216s 1186s 1034w 998m

1294w 1254m 1216s 1186w 1034w 1003s

1291vw 1262vw 1255m

1032w 1003s 409vw

1032vw 1000s 426w

Modea Amide I Amide I Amide I Amide I m(C@C) 8a Amide II Amide II Amide III Amide III Amide III 13 9a 18 12 16b

m, stretching; vs very strong; s, strong; m, medium; w, weak.

details of the spectral and molecular changes for Boc-Gly-X-GlyOME are discussed in [9] while the comparison of characteristic SERS bands for the pairs P2/P20 and P3/P30 are collected in Tables 2 and 3, respectively. Taking into consideration the substitution of the OMe group by the carboxylic group in P20 and P30 , the comparison of the amide I/ II region in FTIR and FT-Raman spectra (Figs. 3–5) shows that secondary structure changes in the couple P2/P20 , whereas this is almost unaffected in the case of the peptides containing D(E)Phe. As discussed in [9], the amide I bands at 1662 and 1640 cm1 are attributed to a-helical and b-turn conformations of P2, whereas two amide I bands at 1666 and 1653 cm1 appear in the spectra of P20 (Fig. 3). The latter suggests that Boc-Gly-D(Z)Phe-fragment remains a a-helical structure, while a folded structure is present in the remaining backbone of the peptide instead of a b-turn. This is indicated by the shift of the amide I band from 1640 (in P2) to 1653 cm1 (in P20 ). Interestingly, such an alternation of the secondary structure is not observed in the D(E)Phe derivatives (P3 and P30 ). Here, the amide I bands assigned to a-helices are found

at 1654 and 1686 cm1 in the IR and Raman spectra of both compounds, while a slight shift of bands originating from b-turns are present at 1629/1695 cm1 in the IR/Raman spectra of P3 and at 1638/1708 cm1 in the spectra of P30 (see [9] and Figs. 4 and 5). Similarly, significant spectral changes corresponding to the m(C@C) mode are found in the FT-Raman spectra of P2 and P20 in the contrast to the couple of P3/P30 . For P2 and P20 , this mode appears at 1623 and 1606 cm1, respectively, whereas its position is found at 1633 and 1638 cm1 in FT-Raman spectra of P3 and P30 , respectively. This finding suggests a significant elongation of the C@C bond in the D(Z)Phe-containing derivatives due to the substitution of the OMe group by the COOH group. In addition as mentioned in [9,17,24], the p-electron conjugation between the phenyl ring and the unsaturated moiety of the peptide backbone can be expected resulting from the great overlap of pC@C and pPh orbitals. This disturbs the electron cloud between C atoms of the C@C bond and the phenyl ring and causes significant changes in the bond polarizabilities. Consequently, the intensity ratio of IC@C/ I8a decreases when a great overlap of pC@C and pPh is induced. We remarked that IC@C/I8a is 0.6 and 1.2 for P2 and P3, respectively [9]. These values were correlated with the torsional angle of the AC@CACPhACPhAmoiety that are ca. 2 and 10 degrees for the -D(Z)Phe-Gly- [25] and -D(E)Phe-Gly- [26] fragments, respectively. Following this assumption, the calculated IC@C/I8a ratios of 0.9 and 1.3 for P20 and P30 , respectively, indicates weakening p-electron conjugation between the phenyl ring and the C@C bond. The significant increase in the IC@C/I8a ratio for P20 in the comparison to P2 may result from the change of the conformation of the peptide and the elongation of the C@C bond, as discussed above. In turn, SERS spectra of the dehydrophenylalanine compounds indicate that the type of the isomer and terminal group strongly affect the adsorption mechanism on the silver electrode. All samples are thermally sensitive, especially the compound P30 , for that two bands typical for a burning product – amorphous carbon, are observed in the SERS spectrum (ca. 1340 and 1560 cm1). Despite this, a few bands appear in the fingerprint region of the SERS spectra. The comparison of the Raman and SERS spectra of the Z isomers (Fig. 3) shows that the SERS profile of the Boc-Gly-XGly-OMe derivative is dominated by the in-plane vibrations of the phenyl ring whereas a contribution of the amide and carboxylate groups into the interaction with the metal is additionally found

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Fig. 5. ATR FTIR spectra of Boc-Gly-DAla-Gly-COOH (P10 ), Boc-Gly-D(Z)Phe-Gly-COOH (P20 ) and Boc-Gly-D(E)Phe-Gly-COOH (P30 ) in the solid state.

for P20 (see Table 2 for details). No broadening of a band assigned to the symmetric stretching mode of the COO group is found in the spectrum of P20 indicating a homogenous way of the adsorption through the p-electron system of this group on the contrary to adsorption of dehydropeptides on silver sols for which significant broadening of this band was observed [17]. The presence of a weak band of the out-of-plane bending vibration of the carboxylate ion indicates that this group lays in a planar orientation on the metal surface [17,23] whereas the lack of the out-of-plane bending motions of the phenyl ring suggests its perpendicular orientation with the respect to the silver. Furthermore, since the positions and fwhm of the peaks do not change considerably due to adsorption on the electrode, a long-distance electromagnetic mechanism of SERS probably plays a dominant role in SERS of P2 and P20 . Interestingly, both molecules do not exhibit an enhancement of the C@C vibrations, observed clearly in the SERS spectra of short peptides [17]. This can result from enforcing a spatial arrangement of dehydrophenylalanine by the flanking Gly residues, which unables a perpendicular orientation of the C@C bond towards the metal surface. Next exchanging the D(Z)Phe moiety by D(E)Phe leads to intensification of the phenyl modes only, c.f. Fig. 4 and Table 3. All modes of the phenyl ring are the in-plane vibrations. For P3, a high intensity of bands assigned to the CC stretching (8a) and breathing (12) modes accompanying by the m(C@C) motion and the 13 mode involving the CbACPh stretching mode indicates the perpendicular orientation of the whole benzylidene group on the metal. While, the P30 molecule only interacts with the silver via the slightly tilted phenyl ring since the in-plane 8a, 12 and outof-plane 16b modes are present in its SERS spectrum. 4. Conclusions In this work, we have highlighted the surface behaviour of the series of six dehydropeptides in terms of surface-enhanced Raman spectroscopy. Secondary structure and the adsorption mechanism on the silver electrode of all peptides are affected by the type of dehydroamino acid residue as well as its type of the isomer. We also noticed that probably weak interactions between this groups of dehydropeptides and the silver electrode result in a variety of SERS profile including its sensitivity to laser exposure on the

contrary to a group of dipeptides containing the same fragment of the peptide backbone. In addition, the presence of different C-terminal groups has an impact on the interaction with the metal. Boc-Gly-DAla-Gly-OMe is not SERS-active molecule whereas its structural analogue possessing the free carboxylic group mainly interacts with the roughened Ag electrode through the amide bonds, although the enhancement of modes of the carboxylate ion is expected. Since Boc-Gly-DAla-Gly-OMe does not provide a SERS signal even with the use of a silver colloid, it seems that the type of the SERS substrate does not affect its SERS activity. Likely, the presence of the alkane bulk groups at the N- and C-end of the molecules prevents from the adsorption on the metal surface. The SERS spectra of tripeptides containing Z and E dehydrophenylalanine significantly differ. The contribution of the peptide backbone along with the planar orientation of the carboxylate ion into the adsorption on the metal surface is found only for the Boc-Gly-D(Z)Phe-Gly-COO species. The SERS spectra of the other tripeptides are dominated by vibrations of the aromatic ring. The comparison of the SERS spectra recorded in this work and those reported in [17] clearly shows that the elongation of the peptide chain in this type of peptides considerably changes their SERS features. Acknowledgement KM and MG would like to thank Dr. Christian Kramberger and the Electronic Properties of Materials group from the Physics Department of the University of Vienna for the access to Raman instrumentation and their assistance with collection of SERS spectra during Erasmus Student-Exchange Programme. We also thank Prof. Jolanta Bukowska and Dr. Agata Krolikowska from the University of Warsaw from their assistance in the preparation of the silver electrodes. This work was financially supported by the Polish Ministry of Science and Higher Education (Grant No. N N204 333037 in 2009–2011). References [1] R. Rink, J. Wierenga, A. Kuipers, L.D. Kluskens, A.J.M. Driessen, O.P. Kuipers, Appl. Environ. Microb. 73 (2007) 1792.

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