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Surface Science 599 (2005) 207–220
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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. The
absence of bands due to amide I for MD2 suggests
that the amide bond lies flat on the surface.
220
E. Podstawka et al. / Surface Science 599 (2005) 207–220
Acknowledgement
Part of this work was supported by internal university grants CRBW/UJ/2003 and WChUJ/DS/
05/2003 (to LMP).
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