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

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Journal of Molecular Structure

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Surface-enhanced Raman spectroscopy of tridehydropeptides adsorbedon silver electrode

http://dx.doi.org/10.1016/j.molstruc.2014.02.0500022-2860/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +48 12 663 2064; fax: +48 12 634 0515.E-mail address: malek@chemia.uj.edu.pl (K. Malek).

Mariusz Gackowski a,b, Kamilla Malek a,⇑a Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Polandb 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

� Peptides containing dehydroalanine andtwo isomers of dehydrophenylalanineare studied.� The structure of the peptides is

investigated by using IR, Raman andSERS techniques.� The adsorption mechanism of the

peptides on the silver electrode isproposed.

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

a r t i c l e i n f o

Article history:Received 14 January 2014Received in revised form 25 February 2014Accepted 25 February 2014Available online 11 March 2014

Keywords:DehydroalanineDehydrophenylalanineTripeptidesSERSAg electrode

a b s t r a c t

Surface-enhanced Raman spectroscopy (SERS) was used to characterise interactions between six tridehy-dropeptides 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 rigiddehydroamino acid residue and the isomer of DPhe have a strong impact on the adsorption mechanismof the peptides. The respective vibrational assignments were proposed by the analysis of FTIR andFT-Raman spectra of solids enabling the evaluation of SERS spectra. Generally, the most intensive SERSbands relative to those in the bulk-phase spectra are associated with vibrations of the dehydroamino acidmoiety, i.e. the C@C bond and the phenyl ring. Only, in the case of the peptides containing the DAla andD(Z)Phe residues and ionised carboxylate group, the molecules interact with the silver electrode via thepeptide backbone. In most cases of the peptide containing DPhe the aromatic ring is almost perpendicularto the metal surface.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Dehydroamino acids are formed by changing sp3 hybridizationof the Ca carbon. The formed double CaCb bond promotes the sterichindrance and imposes conformational restrictions in peptides. Inaddition, this leads to p-electron conjugation between the CaCb

bond and amide groups. This modified amino acid residues havebeen found in a number of naturally occurring peptides like innisins [1] or thiopeptide antibiotics [2] and their action is alsoconnected with biocatalysis [3,4]. The most common in naturedehydroamino acids are dehydroalanine (DAla) and (Z)dehydr-ophenylalanine (D(Z)Phe). The E form of DPhe shows the lowerthermodynamic stability than the Z analogue and is less commonin natural peptides. It is expected that both the isomers possessdifferent conformational structures, and therefore they often serve

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

as pharmacophores [5]. However, details on mechanism of theirpharmacological action have not been yet recognised.

Fourier transform infrared absorption (FTIR) and Raman scat-tering (RS) spectroscopies are well known as a powerful tool instudies on the secondary structure of peptides and proteins inthe solid state and solutions by investigating the spectral amideI, II and III regions sensitive to the peptide backbone structure. Sev-eral experimental [6–11] and theoretical [6,8,11] studies on molec-ular structure of peptides containing DAla and D(E/Z)Phe have beenreported. 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 re-ported up to now [12–16]. However, to our best knowledge, a ten-tative Raman and SERS study of dehydropeptides of variousnumber of dehydroresidues and terminal groups has not been re-ported except of our recent study [17]. In this work we reportedIR, Raman and SERS spectra of three dipeptides Boc-Gly-X, whereX is DAla, D(Z)Phe and D(E)Phe. SERS spectra were collected byusing Ag sol as a metal substrate. In this case, the dehydropep-tide–metal interactions mainly occur due to the deprotonation ofthe terminal carboxylic group. The adsorption process stronglyaffects the appearance of SERS bands in the region of 1500–1650 cm�1, indicating possibility of p-electron resonance betweenthe phenyl ring and the peptide backbone. SERS technique was alsoused in studies on pentapeptides containing DPhe [18]. This inves-tigation showed that this type of peptides is promising a cappingagent for gold nanoparticles exhibiting properties of drug deliveryvehicles.

In the present study, we focus on an evaluation of SERS featuresof tridehydropeptides, in which a rigid central moiety (DAla, DPhe)is surrounded by two flexible glycine residues. The following pep-tides 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-Gly-OMe (P3) (Boc, t-butoxycarbonyl; OMe, methoxy), see Scheme 1.In addition, we investigate SERS profile of their structural analo-gous, in which the OMe group is substituted by the COOH group(P10, P20, and P30, respectively) since it is well known that thisgroup exhibits the high affinity to the metal surface [17,23].

Scheme 1. Structures of the tripeptides P1–P3.

Despite this the chosen molecules exhibit a variety of conforma-tional preferences in the solid state that may be reflected in theirability to adsorb on the metallic support. Thus, we also discuss FTIRand Raman profile of the solids to give an insight into molecularstructures of the peptides. The main aim of this work is to deter-mine the nature of the interaction of these peptides with the solidsilver surface as well as to determine how the type of dehydroami-no acid residue and the C-terminal functional group affect peptideability to adsorb on the silver electrode.

2. Experimental

Compounds were synthesized according to the procedure de-scribed in [19]. Briefly, peptides were synthesized in condensationreaction between trifluroacetate (TFA) amide of alanine or phenyl-alanine and a-keto acid (pyruvic acid or phenylpyruvic acid) in ben-zene. The reaction is catalysed by p-toluenesulfonic acid. In the caseof TFA-Gly-DPhe-Gly, both isomers (Z and E) are formed in ratio of4:1. Then, the TFA group is substituted by the Boc group. Yield for allsyntheses: 50–80%. The purity of the compounds were tested bystandard analytical methods (elemental analysis and NMR).

For FT-Raman measurements, a few milligrams of each solidsample were measured on metal discs directly. Spectra wereaccumulated from 256 scans, with a spectral resolution of4 cm�1. Spectra were recorded on a MultiRAM FT-Raman Spec-trometer (Bruker), equipped with a germanium detector cooledwith liquid nitrogen. Each of the samples was illuminated from aNd:YAG laser (k = 1064 nm) at an output power of 100 mW. ATRFTIR spectra of the solid samples were collected using a ALPHABruker spectrometer equipped with a 1-reflection ATR diamondcrystal. Spectra were collected in the range of 375–4000 cm�1,with spectral resolution of 4 cm�1. 128 scans were co-added, andthen extended ATR correction was employed.

For SERS measurements, solids were dissolved in ethanol/waterin volume ratio of 1:1 to prepare 1 � 10�3 M solutions. After dis-solving pH of solutions was ca. 6. Since, protonation constant ofthe COOH group in dehydropeptides is found in the range of 3–4,this group should be deprotonated in P10–P30 during collection ofSERS spectra [20]. The silver electrodes were prepared by electro-chemical roughening, using five positive/negative cycles in 0.1 MKCl solution from �0.3 to +0.3 V (vs Ag/AgCl) and the potentialclose to the end of the last negative cycle was held for 30 s. A plat-inum electrode was used as a counter electrode while Ag/AgCl in1 M KCl was used as a reference. The silver electrodes were im-mersed in 1 � 10�3 M solutions of each peptide, then SERS spectrawere recorded after a few hours since no SERS signal was observedimmediately after immersing the electrode in the solution. FromSEM pictures one can deduce that the electrode is not roughenedequally on its whole surface (Fig. 1A and B). Roughness of the silversurface also varies in thickness. The SERS measurements were con-ducted on a LabRam 800 Raman Spectrometer equipped in a con-focal microscope, a CCD detector and a He–Ne laser excitationline (632.8 nm). The spectra were collected through an air objec-tive with the magnification of 50� with the use of 600 g/mm grat-ing. For each SERS spectrum 60 scans were collected withintegration time of 1 s. Output laser power was set from 0.154 to3.4 mW. Laser power for each measurement of SERS spectrumwas adjusted in such a way to use a minimal power and to obtainspectrum showing a good signal to noise ratio.

3. Results and discussion

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

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

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

M. Gackowski, K. Malek / Journal of Molecular Structure 1065-1066 (2014) 143–149 145

In the case of Boc-Gly-DAla-Gly-OMe (P1), SERS spectra showeddifferent spectral profiles during measurements from variouspoints of the electrode exhibiting likely photodegradation processof the peptide due to excitation by laser light (data not shown). It isworth-mentioning that we attempted to collect SERS spectra of P1on silver colloids and laser excitations in the Vis and NIR region,but no SERS signal or large variability of signal were observed dur-ing measurements. The selected band’s positions in FT-Raman andSERS spectra with their tentative assignments are collected inTables 1–3 [9,12–17,21,22]. Here, we assign only major peaks,which appear in SERS spectra, to give an insight into the adsorptionmechanism of the studied dehydropetides. FTIR and FT-Ramanspectra of P1–P3 are discussed in detail in [9], while ATR FTIR spec-tra 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 cm�1 (typical for the amide Iand C@C vibrations), indicates significant differences in a second-ary structure of both compounds (c.f. Fig. 2). A detailed examina-tion of FTIR and FT-Raman spectra of P1 exhibited spectralmarkers specific for an extended conformation of the peptide con-firmed by its crystallographic structure [9]. Amide I bands were ob-served at 1690 and 1660 cm�1 in IR spectrum of P1 [9], whereasthree IR bands at 1677, 1656, and 1632 cm�1 are present in theATR FTIR spectrum of P10 (Fig. 5). The latter indicates that due tothe substitution of the OMe group in P1 by the COOH group inP10, the tridehydropeptide P10 adopts a folded structure. This is alsoconfirmed by the IR position of the amide II band at 1504 cm�1

accompanied by a few shoulders at 1560 and 1537 cm�1 (c.f.Fig. 5). The appearance of a large number of amide II bands canbe associated with variation in dihedral angles in the peptide

backbone. In turn, SERS spectra of both peptides suggest an impactof this substitution on interactions between the metal and thedehydropeptides containing dehydroalanine. As mentioned above,P1 adsorbed on the silver electrode is probably decomposed duringexposure to laser light, whereas a good quality SERS spectrum wasrecorded for P10 (see Fig. 2). Vibrations assigned to SERS bands ofP10 are collected in Table 1.

The principal bands in the SERS spectrum of P10 are those corre-sponding to the amide II and amide III vibrations, the symmetricstretches of the deprotonated carboxylic group, and the bendingmodes of the glycine residues. The amide II and amide III are quitedistinctive, signifying that the amide groups of the peptide are notperpendicular to the silver surface since both are the combinationof the stretching and bending vibrations. The presence of thesebands were also identified in SERS spectrum of the P10 analoguewith a shorter backbone, i.e. Boc-Gly-DAla-COOH [17]. Howeverthe amide I mode was more intensified than other amide vibra-tions in SERS of this dehydropeptide on the contrary to P10. Thiscan indicate that the amide moieties are tilted with respect tothe metal electrode. The observed shift of the SERS amide bandsfrom their counterparts in the FT-Raman spectrum of the solidsample (15–20 cm�1) may in addition suggest some changes inthe secondary structure of P10. According to the previous studies[6,9,17], the high-wavenumber amide II bands are specific for aa-helical structure typical for the Boc-Gly-moiety, while a bandat 1508 cm�1 appears due to a turn conformation around thedehydroamino acid residue (DAla). The bands at 1581 and1547 cm�1 exhibit a higher SERS intensity than the 1508 cm�1

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

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

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

ATR FTIR FT Raman SERS Modea

1677m 1678vs Amide I1656vs 1658m,sh Amide I1632m 1628m Amide I1560w,sh 1559vw 1581s Amide II1537m,sh 1532vw 1547s Amide II1504vs 1510w 1508s Amide II

1454m 1448m d(CH2)Gly

1418s 1411m 1421s d(@CH2)1370s 1375vw 1379m ms(COO�), x(CH2)Gly

1322w 1326vs Amide III1260m 1263s 1252m Amide III

1247m,sh 1231m s(CH2)Gly

1093m 1107m m(CN), mas(COC)Boc

1062m 1059s 1074m q(CH3)Boc, x(C-O)Boc

678m 681vw 676m 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.

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

band (Fig. 2). We interpret the relative intensity of these bands asan evidence that the modes of a a-helical conformation have com-ponents of Raman polarizability perpendicular to the metal sur-face. Since, the largest shift is found for the bands assigned tothe helical structure, likely the conformation of the Boc-Gly-frag-ment of P10 changes due to the adsorption on the silver. This is alsoconfirmed by the 9 cm�1 red-shift of the amide III band at1252 cm�1 attributed to helical structures [9]. The other featureof the SERS spectrum of Boc-Gly-DAla-COOH is an enhanced bandof the >C@CH2 stretching mode at 1621 cm�1 [17], however, thisband is not present in the SERS spectrum of Boc-Gly-DAla-Gly-COOH. For the latter, we only observe the intensification of thescissoring mode of the @CH2 group at 1421 cm�1, suggesting thatblocking the dehydro moiety by the second Gly residue preventsfrom a perpendicular orientation of the DAla group on theelectrode. In addition, the deprotonated C-terminal group oftenparticipates in the adsorption on the metal through its delocalizedp-electron system [17,23]. This is verified by the presence of a SERSband in the region of 1360–1420 cm�1 that is attributed to thesymmetric stretching vibration of the COO� group. In the SERSspectrum of P10, we assign a SERS band at 1379 cm�1 of a mediumintensity to this mode. Since, the protonated molecules of thedehydropeptide are present in solution (pH �6) used in SERS mea-surements, the appearance of this band indicates the removal ofthe carboxylic proton due to the interaction with the metal.According to Fleger and co-workers [23], the orientation of the car-boxylic group can be identified by relative intensity of SERS bandsassigned to the stretching and bending vibrations of the COO�

group. For P10, both bands at 1379 and 676 cm�1, respectively,

exhibit similar intensity, thus the carboxylate anion adopts a bentorientation preventing a direct interaction of its delocalised p-elec-tron system with the metal surface.

The introduction of the bulk dehydrophenylalanine between theglycine residues leads to completely different IR, Raman and SERSfeatures in the comparison to the DAla moiety (see Figs. 2–5). Thechange in the type of the isomer from Z to E also affects molecularstructure of a peptide, and consequently vibrational spectra. The

Table 2Positions of ATR FTIR, FT-Raman, and SERS bands (in cm�1) 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].

P2 P20

ATR FTIR FT-Raman SERS ATR FTIR FT-Raman SERS Modea

1685s 1688m Amide I1656vs 1662s 1664s 1666m Amide I1640m,sh 1640s 1650vs 1653s Amide I1620m 1623vs 1606vs m(C@C)

1604vs 1598s 1601vs 1599vs 1596vs 8a1558m,sh 1565m,sh 1564m Amide II

1523s 1528vw 1537m Amide II1493s 1478s Amide II

1457w 1453vw 1468w d(CH3)Boc

1443m 1448w 1445m d(CH2)Gly

1362s ms(COO�)1353s

1321w 1328m 1314vw Amide III1252m,sh 1255w 1281m 1290w Amide III1210s 1211vs 1206w 1207s 1206vs 1200m 13

1120w 1101w 1101vw 1102m m(CN), mas(COC)Boc

1001s 1003m 1003s 1000m 12838w 844vw 837w m(CC), x(COO�)655s 654w 5

a 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 3Positions of ATR FTIR, FT-Raman, and SERS bands (in cm�1) of Boc-Gly-D(E)Phe-Gly-OMe (P3) and Boc-Gly-D(E)Phe-Gly-COOH (P30) together with the proposed assign-ment [9,12–17,21,22].

P3 P30

ATR FTIR FT-Raman SERS ATR FTIR FT-Raman SERS Modea

1695vs 1708 Amide I1686m Amide I

1655vs 1654w 1654s Amide I1629s Amide I

1633vs 1636w 1638vs m(C@C)1600vs 1602s 1601s 1601s 8a

1543s 1533vs Amide II1511vs Amide II1301m 1304m 1294w 1291vw Amide III1268m 1268vw 1262vw Amide III1252s 1255vw 1254m 1255m Amide III

1216s 1216s 131186s 1186w 9a1034w 1034w 1032w 1032vw 18998m 1003s 1003s 1000s 12

409vw 426w 16b

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

M. Gackowski, K. Malek / Journal of Molecular Structure 1065-1066 (2014) 143–149 147

details of the spectral and molecular changes for Boc-Gly-X-Gly-OME are discussed in [9] while the comparison of characteristicSERS bands for the pairs P2/P20 and P3/P30 are collected in Tables2 and 3, respectively.

Taking into consideration the substitution of the OMe group bythe 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 sec-ondary structure changes in the couple P2/P20, whereas this is al-most unaffected in the case of the peptides containing D(E)Phe.As discussed in [9], the amide I bands at 1662 and 1640 cm�1 areattributed to a-helical and b-turn conformations of P2, whereastwo amide I bands at 1666 and 1653 cm�1 appear in the spectraof P20 (Fig. 3). The latter suggests that Boc-Gly-D(Z)Phe-fragmentremains a a-helical structure, while a folded structure is presentin the remaining backbone of the peptide instead of a b-turn. Thisis indicated by the shift of the amide I band from 1640 (in P2) to1653 cm�1 (in P20). Interestingly, such an alternation of the sec-ondary structure is not observed in the D(E)Phe derivatives (P3and P30). Here, the amide I bands assigned to a-helices are found

at 1654 and 1686 cm�1 in the IR and Raman spectra of both com-pounds, while a slight shift of bands originating from b-turns arepresent at 1629/1695 cm�1 in the IR/Raman spectra of P3 and at1638/1708 cm�1 in the spectra of P30 (see [9] and Figs. 4 and 5).

Similarly, significant spectral changes corresponding to them(C@C) mode are found in the FT-Raman spectra of P2 and P20 inthe contrast to the couple of P3/P30. For P2 and P20, this mode ap-pears at 1623 and 1606 cm�1, respectively, whereas its position isfound at 1633 and 1638 cm�1 in FT-Raman spectra of P3 and P30,respectively. This finding suggests a significant elongation of theC@C bond in the D(Z)Phe-containing derivatives due to the substi-tution of the OMe group by the COOH group. In addition as men-tioned in [9,17,24], the p-electron conjugation between thephenyl ring and the unsaturated moiety of the peptide backbonecan be expected resulting from the great overlap of pC@C and pPh

orbitals. This disturbs the electron cloud between C atoms of theC@C bond and the phenyl ring and causes significant changes inthe bond polarizabilities. Consequently, the intensity ratio of IC@C/I8a decreases when a great overlap of pC@C and pPh is induced. Weremarked 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 theAC@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 0.9and 1.3 for P20 and P30, respectively, indicates weakening p-elec-tron conjugation between the phenyl ring and the C@C bond. Thesignificant increase in the IC@C/I8a ratio for P20 in the comparisonto P2 may result from the change of the conformation of the pep-tide and the elongation of the C@C bond, as discussed above.

In turn, SERS spectra of the dehydrophenylalanine compoundsindicate that the type of the isomer and terminal group strongly af-fect the adsorption mechanism on the silver electrode. All samplesare thermally sensitive, especially the compound P30, for that twobands typical for a burning product – amorphous carbon, are ob-served in the SERS spectrum (ca. 1340 and 1560 cm�1). Despitethis, a few bands appear in the fingerprint region of the SERSspectra. The comparison of the Raman and SERS spectra of the Zisomers (Fig. 3) shows that the SERS profile of the Boc-Gly-X-Gly-OMe derivative is dominated by the in-plane vibrations ofthe phenyl ring whereas a contribution of the amide and carboxyl-ate groups into the interaction with the metal is additionally found

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.

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

for P20 (see Table 2 for details). No broadening of a band assigned tothe symmetric stretching mode of the COO� group is found in thespectrum of P20 indicating a homogenous way of the adsorptionthrough the p-electron system of this group on the contrary toadsorption of dehydropeptides on silver sols for which significantbroadening of this band was observed [17]. The presence of a weakband of the out-of-plane bending vibration of the carboxylate ionindicates that this group lays in a planar orientation on the metalsurface [17,23] whereas the lack of the out-of-plane bending mo-tions of the phenyl ring suggests its perpendicular orientation withthe respect to the silver. Furthermore, since the positions andfwhm of the peaks do not change considerably due to adsorptionon the electrode, a long-distance electromagnetic mechanism ofSERS probably plays a dominant role in SERS of P2 and P20. Inter-estingly, both molecules do not exhibit an enhancement of theC@C vibrations, observed clearly in the SERS spectra of short pep-tides [17]. This can result from enforcing a spatial arrangement ofdehydrophenylalanine by the flanking Gly residues, which unablesa perpendicular orientation of the C@C bond towards the metalsurface. Next exchanging the D(Z)Phe moiety by D(E)Phe leads tointensification 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, ahigh intensity of bands assigned to the CC stretching (8a) andbreathing (12) modes accompanying by the m(C@C) motion andthe 13 mode involving the CbACPh stretching mode indicates theperpendicular orientation of the whole benzylidene group on themetal. While, the P30 molecule only interacts with the silver viathe slightly tilted phenyl ring since the in-plane 8a, 12 and out-of-plane 16b modes are present in its SERS spectrum.

4. Conclusions

In this work, we have highlighted the surface behaviour of theseries of six dehydropeptides in terms of surface-enhanced Ramanspectroscopy. Secondary structure and the adsorption mechanismon the silver electrode of all peptides are affected by the type ofdehydroamino acid residue as well as its type of the isomer. Wealso noticed that probably weak interactions between this groupsof dehydropeptides and the silver electrode result in a variety ofSERS profile including its sensitivity to laser exposure on the

contrary to a group of dipeptides containing the same fragmentof the peptide backbone. In addition, the presence of differentC-terminal groups has an impact on the interaction with the metal.Boc-Gly-DAla-Gly-OMe is not SERS-active molecule whereas itsstructural analogue possessing the free carboxylic group mainlyinteracts with the roughened Ag electrode through the amidebonds, although the enhancement of modes of the carboxylateion is expected. Since Boc-Gly-DAla-Gly-OMe does not provide aSERS signal even with the use of a silver colloid, it seems thatthe type of the SERS substrate does not affect its SERS activity.Likely, the presence of the alkane bulk groups at the N- andC-end of the molecules prevents from the adsorption on the metalsurface. The SERS spectra of tripeptides containing Z and E dehydr-ophenylalanine significantly differ. The contribution of the peptidebackbone along with the planar orientation of the carboxylate ioninto the adsorption on the metal surface is found only for theBoc-Gly-D(Z)Phe-Gly-COO� species. The SERS spectra of the othertripeptides are dominated by vibrations of the aromatic ring. Thecomparison of the SERS spectra recorded in this work and those re-ported in [17] clearly shows that the elongation of the peptidechain in this type of peptides considerably changes their SERSfeatures.

Acknowledgement

KM and MG would like to thank Dr. Christian Kramberger andthe Electronic Properties of Materials group from the PhysicsDepartment of the University of Vienna for the access to Ramaninstrumentation and their assistance with collection of SERSspectra during Erasmus Student-Exchange Programme. We alsothank Prof. Jolanta Bukowska and Dr. Agata Krolikowska from theUniversity of Warsaw from their assistance in the preparation ofthe silver electrodes. This work was financially supported by thePolish Ministry of Science and Higher Education (Grant No.N N204 333037 in 2009–2011).

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