# Interaction of Bradykinin and B2 Bradykinin Receptor Antagonists with Colloidal Au Surface Explored by Surface-Enhanced Raman Scattering

Authors: Dominika Skołuba; Dariusz Sobolewski; Adam Prahl; Edyta Proniewicz
Journal: Journal of Spectroscopy
 (2014)

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## Abstract

Bradykinin (BK), an endogenous peptide hormone, which is involved in a number of physiological and pathophysiological processes, and the potent B2 bradykinin receptor antagonists, [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, were deposited onto colloidal Au particles of 20 nm size. Interaction of these molecules with colloidal Au surface was explored by surface-enhanced Raman scattering (SERS). Briefly, it was shown that BK adsorbs on the Au surface mainly through the Phe5/Phe8 residues. In case of the BK specifically modified analogues mainly the Pip and Thi rings are involved in the interaction process; however, Pip and Thi adopt slightly different orientation with respect to Au for each of the analogues. In addition, the lack of the Aaa vibrations, together with the enhancement of the Thi, Pip, or Phe modes, emphasizes the importance of the C-terminus in the interaction with the Au surface.


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## Body

## 1. Introduction

Bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is an important endogenous oligopeptide counted from the family of kinins [1]. Biological activities of BK are associated with a number of physiological and pathophysiological processes, including regulation of vascular permeability, formation of edema, stimulation of nerve endings, and inflammation process [2, 3]. It is also a potent cancers growth factor, especially prostate and lung carcinoma [4, 5]. Biological effects of BK are mediated by activation of the G protein coupled seven transmembrane receptors (GPCRs), named B1 and B2 [6]. GPCRs are the largest group of the membrane receptors [7]. The B1 receptors are poorly detectable under physiological conditions, whereas the B2 receptors are continuously produced. The B2 receptors mediate most of the biological effects and demonstrate a high affinity for BK [8].The biological significance of BK motivated researchers to explore behavior of this peptide at different solid/liquid interfaces. For example, studies using nuclear magnetic resonance (NMR) [9, 10], circular dichroism (CD) [11], and molecular dynamics [12] have explored mode of BK binding to different membranes and pointed out the formation of the BK C-terminal (Ser6-Arg9) β-turn structure in the membrane-bound state of BK that is crucial for this binding to the B2 receptor [12, 13]. Studies on BK adsorbed onto the electrochemically roughened Ag, Au, and Cu electrode surfaces in an aqueous solution at physiological pH at different applied electrode potential have shown that BK interacts mainly through the two Phe (L-phenylalanine), Arg (L-arginine), and Pro (L-proline) residues, but in different arrangements, with each of the metal surfaces [14]. These results are similar to those from the biological activity studies [15]. Also, experiments for isotopically labeled BK deposited onto the Ag electrode have demonstrated that the reorientation of the BK phenyl rings cannot keep up with the electrode potential changes at the electrode potential range from −0.8 to −0.4 V [16]. Another examination of four of the B2 BK receptor antagonists immobilized onto Ag SERS-active: ROC (prepared in the reduction-oxidation cycles) and colloidal substrates have demonstrated that the adsorption mode of BK varied in time on the ROC substrate and the ROC surface is more selective than the colloidal one [17, 18].In this work, using surface-enhanced Raman spectroscopy (SERS) we characterized adsorption mode of BK and its four synthetic B2 BK receptor antagonists: [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK (where Aaa denotes 1-adamantaneacetic acid, Hyp—L-hydroxyproline, Thi—L-thienylalanine, and Pip—L-pipecolic acid) onto colloidal Au spheres with the diameters of approximately 20 nm. The SERS technique was earlier used in our laboratory to correlate the structural components of the many different peptides, including bombesin, vasopressin, neurotensin, and their modified analogs with their capability to interact with the proper metabotropic receptor [19–22]. Also, our aim is correlation of information resulting from the spectroscopic and biological activity investigations.Since 1979, it is known that the composition of the metallic nanostructures has influence on the intensity of the SERS signal. Ag enhances the Raman signal more (10–100-fold) than Au because of the plasmon resonance [23]. In addition, Ag can be excited in the light range from the UV to the IR, whereas Au is limited to the red or IR due to damping by the interband transitions [24]. Despite these limitations, essential structural studies for living organisms are commonly performed for Au. It is due to significantly higher biocompatibility and better control of the size and shape of Au in comparison to the Ag nanostructures [25].


## 2. Materials and Methods

### 2.1. Peptide Synthesis

BK, [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK were obtained by the solid-phase method using the Fmoc-strategy starting from Fmoc-Arg(Pbf)-Wang resin (GL Biochem Shanghai Ltd., 1% DVB, 100–200 mesh, 0.47 mmol/g) [26]. Fmoc was removed by 20% piperidine in DMF. A 3-fold excess of the respective Fmoc-amino acids was activated in situ using HATU (1 eq)/HOAt (1 eq) in a mixture of DMF/NMP (1 : 1 v/v) containing 1% Triton, and the coupling reactions were base-catalyzed with NMM (2 eq). The amino acid side-chain-protecting groups were Bu
t for Hyp and Ser and Pbf for Arg and D-Arg. All of the Fmoc-protected amino acids were commercially available (NovaBiochem, Bad Soden, Germany). Aaa (1-adamantaneacetic acid) was coupled in the final coupling step (for acylated peptides) using the same procedure as that for Fmoc-amino acids. Cleavage of the peptides from the resin with side-chain deprotection was performed by treatment with TFA : H2O : TIS (95 : 2.5 : 2.5 v/v/v) for 4 h. The total volume of the TFA filtrate was reduced to approximately 1 mL by evaporation in vacuo. The peptides were precipitated with cold diethyl ether and filtered through a Schott funnel. All of the peptides were purified by semipreparative high-performance liquid chromatography (HPLC). HPLC was executed on a Waters (analytical and semipreparative) chromatograph equipped with a UV detector (λ = 226 nm). The purity of the peptides was determined on a Discovery HS C18 column (5 μm, 100 Å; 250 × 4.6 mm). The solvent systems were (A) 0.1% aqueous trifluoroacetic acid (TFA) and (B) 80% acetonitrile in aqueous 0.1% TFA (v/v). A linear gradient from 1 to 80% of (B) over 30 min was applied for peptides at a flow rate of 1 mL/min. Semipreparative HPLC was performed using a Kromasil C8 column (5 μm, 100 Å; 16 × 250 mm) in a linear gradient from 10 to 40% of (B) for 90 min at a flow rate of 8 mL/min. The mass spectra of the peptides were recorded on a Bruker BIFLEX III MALDI TOF mass spectrometer (ionization: 337 nm nitrogen laser).


### 2.2. SERS Measurement in Au Colloid Solution

The Au colloid was purchased from Sigma Aldrich (stabilized suspension in citrate buffer, diameter 20 nm). Aqueous solution of the peptides was prepared by dissolution of the peptides in deionized water. The concentration of the peptide solutions was adjusted to10
-
4 M before them being mixed with the Au colloid. 10 μL of peptide sample was mixed with 20 μL of Au nanoparticles. The SERS spectra of the peptides were obtained using Renishaw spectrometer (model inVia) operating in confocal mode combined with a Peltier cooled CCD detector and a Leica microscope (50x long-distance objective). Excitation wavelength at 785 nm was used from HP NIR diode laser. The laser power at the laser output was set at approximately 20 mW. Generally, the SERS spectra were measured at four spots on the surfaces of the colloidal Au nanoparticles and were obtained during 2 h of sample addition to the Au solution. The spectra from the series were nearby identical, except for small differences in some band intensities. During measurements no spectral changes due to the sample decomposition or desorption processes were observed.


### 2.3. Spectral Analysis

The spectral analysis was performed using the GRAMS/AI 8.0 (Thermo Electron Corp.).


## 2.1. Peptide Synthesis

BK, [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK were obtained by the solid-phase method using the Fmoc-strategy starting from Fmoc-Arg(Pbf)-Wang resin (GL Biochem Shanghai Ltd., 1% DVB, 100–200 mesh, 0.47 mmol/g) [26]. Fmoc was removed by 20% piperidine in DMF. A 3-fold excess of the respective Fmoc-amino acids was activated in situ using HATU (1 eq)/HOAt (1 eq) in a mixture of DMF/NMP (1 : 1 v/v) containing 1% Triton, and the coupling reactions were base-catalyzed with NMM (2 eq). The amino acid side-chain-protecting groups were Bu
t for Hyp and Ser and Pbf for Arg and D-Arg. All of the Fmoc-protected amino acids were commercially available (NovaBiochem, Bad Soden, Germany). Aaa (1-adamantaneacetic acid) was coupled in the final coupling step (for acylated peptides) using the same procedure as that for Fmoc-amino acids. Cleavage of the peptides from the resin with side-chain deprotection was performed by treatment with TFA : H2O : TIS (95 : 2.5 : 2.5 v/v/v) for 4 h. The total volume of the TFA filtrate was reduced to approximately 1 mL by evaporation in vacuo. The peptides were precipitated with cold diethyl ether and filtered through a Schott funnel. All of the peptides were purified by semipreparative high-performance liquid chromatography (HPLC). HPLC was executed on a Waters (analytical and semipreparative) chromatograph equipped with a UV detector (λ = 226 nm). The purity of the peptides was determined on a Discovery HS C18 column (5 μm, 100 Å; 250 × 4.6 mm). The solvent systems were (A) 0.1% aqueous trifluoroacetic acid (TFA) and (B) 80% acetonitrile in aqueous 0.1% TFA (v/v). A linear gradient from 1 to 80% of (B) over 30 min was applied for peptides at a flow rate of 1 mL/min. Semipreparative HPLC was performed using a Kromasil C8 column (5 μm, 100 Å; 16 × 250 mm) in a linear gradient from 10 to 40% of (B) for 90 min at a flow rate of 8 mL/min. The mass spectra of the peptides were recorded on a Bruker BIFLEX III MALDI TOF mass spectrometer (ionization: 337 nm nitrogen laser).


## 2.2. SERS Measurement in Au Colloid Solution

The Au colloid was purchased from Sigma Aldrich (stabilized suspension in citrate buffer, diameter 20 nm). Aqueous solution of the peptides was prepared by dissolution of the peptides in deionized water. The concentration of the peptide solutions was adjusted to10
-
4 M before them being mixed with the Au colloid. 10 μL of peptide sample was mixed with 20 μL of Au nanoparticles. The SERS spectra of the peptides were obtained using Renishaw spectrometer (model inVia) operating in confocal mode combined with a Peltier cooled CCD detector and a Leica microscope (50x long-distance objective). Excitation wavelength at 785 nm was used from HP NIR diode laser. The laser power at the laser output was set at approximately 20 mW. Generally, the SERS spectra were measured at four spots on the surfaces of the colloidal Au nanoparticles and were obtained during 2 h of sample addition to the Au solution. The spectra from the series were nearby identical, except for small differences in some band intensities. During measurements no spectral changes due to the sample decomposition or desorption processes were observed.


## 2.3. Spectral Analysis

The spectral analysis was performed using the GRAMS/AI 8.0 (Thermo Electron Corp.).


## 3. Results and Discussion

Figure1 presents the SERS spectra of Figure 1(a) BK and its four potent B2 BK receptor antagonists: Figure 1(b) [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Figure 1(c) Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Figure 1(d) [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Figure 1(e) Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK (see Table 1 for molecular structures) in the Au aqueous solution excited with 785 nm line. The spectral positions of the enhanced bands in these spectra, given in wavenumbers, are summarized in Table 2 together with the proposed vibrational assignments. This assignment leans on the earlier studies of amino acids lyophilized and deposited onto the colloidal Au surface [27, 28], BK, and its modified analogs [16–18], piperidine (Pip) [29, 30] and thiophene (Thi) [31–33]. Based on the analysis of the changes in the bands enhancement, bands width, and shift in bands wavenumber between corresponding Raman [14, 16–18] and SERS spectra conclusions about the adsorption process of BK and its four analogs deposited onto the colloidal Au surface were drawn as described below.Table 1
Amino acid sequence of bradykinin (BK) and its specifically mutated analogues investigated in this work.Table 2
Wavenumbers and proposed band assignments for SERS spectra of A: BK, B: [D-Ar
g
0,Hy
p
3,Th
i
5,8,L-Pi
p
7]BK, C: Aaa[D-Ar
g
0,Hy
p
3,Th
i
5,8,L-Pi
p
7]BK, D: [D-Ar
g
0,Hy
p
3,Th
i
5,8,D-Ph
e
7,L-Pi
p
8]BK, and E: Aaa[D-Ar
g
0,Hy
p
3,Th
i
5,D-Ph
e
7,L-Pi
p
8]BK adsorbed onto colloidal gold surface.
Wavenumber/c
m
-
1
A
B
C
D
E
ρ
s(NH2), Arg[ν
s(C=N)], and/or Amide I
—
1631
1615
1639
—
Phe[ν
8a] and/or Arg[δ(NH
2
+) + ν
s(C=N)]
1603
—
—
1608
1602
Phe[ν
8b] and/or Pip and/or Arg[δ(NH)]
1585
1579
1596
—
1570
Pip
—
—
1551
—
—
Imide II’ and/or Thi[ν(CC)]
1528
1521
—
—
—
Thi[ν(C–C)]
—
1500
1495
1504
1501
Phe[ν
19b], Arg[δ(NH)], and/or tertiary amide IIp marker
1443
1425
1434
1438
1438
Thi[δ(CH) + ν(C=C)], Pip combination, and/or ν(COO−)
—
1365
1379
1360
1377
Arg[ρ
sb(CH3)]
1341
—
1339
—
—
Pip[ρ
w(CH2)]
—
1310
—
—
—
Pip[ν(CN) + ρ
b(CCH) + ρ
r/t(CH2)] and/or Amide III
—
1288
1281
1284
1275
Phe[ν
4]
1266
—
—
—
—
Pip[ρ
t(CH2)] and/or Amide III
—
—
1242
1251
—
Phe[ν
7a]
1203
—
—
—
—
Thi[ν(CH)]
—
1204
1215
1202
1212
Arg[ν(CN)] and/or Thi[ρ
r(CH)]
—
1170
1179
1165
1181
Arg[ν(CCC)] and/or Pip[ρ
t(CH2)]
—
1148
1142
1151
1144
Aaa[ν(CC)]
—
—
1111
—
1102
Thi[in-planeρ
r(CH)] and/or Arg [ν(CN)]
—
—
—
1093
—
Arg[ν(CN)] and/or Thi[ρ
r(CH)]
—
1083
1070
—
1083
Arg[ν(CC)  +  ν(NC)]
1058
—
—
1045
Phe[ν
18a]
1031
—
—
1026
Pip[skeletal stretching]
—
1020996
1019
1013
Phe[ν
12]
1002
—
—
1000
—
ν(CC) + ρ
r(CH2)
958
—
945
957
—
—
—
—
939
936
Arg[ρ
b(NH2)], Pip[ρ
r(CH)], and/or Thi[ρ
w(CH2) + γ(ring)]
—
—
—
925
Thi[δ(ring) + ν(CS)] and/or Pip skeletal stretching
—
—
881
887
—
Arg[δ(NH)], Thi[δ(ring)  +  ν(CC)], and/or Pip ν(CC)
840
850
—
840
—
Pip ring breathing, Arg[ρ
w(NH2)], and/or Thi[ν(CC)  +  δ(ring)]
—
812
833
839
Pip[ring breathing]
—
776
763
763
752
Pro[ρ
r(CH2)ring]
765
—
—
—
Pip
—
723
—
729
725
Pip
—
—
717
700
Thi[ρ
w(CH)] and/or  Arg [δ(COO−)]
681
678
671
682
670
ρ
b(COO) and/or Amide
—
—
652
—
—
Phe[ν
6b]
620
—
—
—
—
Pip
—
612
—
594
—
Thi[γ(ring) + ρ
w(CH2)]
—
—
568
—
—
ρ
b(CO) and/or ρ
r(CH2)
—
538
—
534
—
Pip
—
499
—
—
—
Skeletal
467
—
464
—
457
Thi[γ(ring)]
—
—
426
—
—
ν: stretching, δ: deformation, γ: torsion, ρ
t: twisting, ρ
b: bending ρ
r: rocking, ρ
w: wagging, and ρ
s: scissoring vibrations; Phe: phenylalanine, Arg: arginine, Thi: L-thienylalanine, Aaa: 1-adamantaneacetic acid, and Pip: L-pipecolic acid.Figure 1
The SERS spectra of (a) BK and its analogues: (b) [D-Arg0,Hyp3,Thi5,8,L-Pip7] BK, (c) Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, (d) [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and (e) Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK adsorbed onto a colloidal Au particles surface in an aqueous solution.The SERS spectrum of BK (Figure1(a)) is dominated by 1603 [ν
8a], 1585 [ν
8b], 1443 [ν
19b], 1203 [ν
7a], 1031 [ν
18a], 1002 [ν
12], and 620 cm−1 [ν
6b] bands due to the Phe residues (Phe5/Phe8) vibrations. According to the so-called surface selection rules, the phenyl ring orientation with respect to the colloidal Au substrate could be predicted based on the relative intensity of the aforementioned bands [34, 35]. For a perpendicular orientation of an aromatic ring onto metal surface these rules postulate that the in-plane modes should be mainly enhanced in the SERS spectra. On the other hand, for a horizontal orientation of the aromatic ring on the metal surface the out-of-plane vibrations should be stronger than the in-plane vibrations. Thus, based on the propensity rules and phenomenon that the in-plane 1002 cm−1 band is the strongest band in the BK SERS spectrum and is less intense than that in the BK Raman spectrum it can be suggested that the BK phenyl rings adopt the tilted orientation with respect to the Au nanoparticles surface [14, 16]. The neglected shift in wavenumber and 4 cm−1 band broadening of the 1002 cm−1 SERS signal in comparison with those in the corresponding Raman spectrum together with the symmetric shape of this band indicate that the BK phenyls rings do not interact directly with Au. Thus, should be located in slight distance from Au.Apart from the phenyl rings, the Arg and Pro residues are also involved in the interaction of BK with the colloidal Au surface. The Arg and Pro oscillations at 1443 [δ(NH)], 1341 [ρ
sb (CH3)], 1058 [ν(CC) + ν(NC)], and 840 [δ(NH)] cm−1 and at 1528, 1443, and 765 cm−1 (see Table 2 for the bands assignment), respectively, determine this interaction.In the SERS spectrum of [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK (Figure 1(b)) the weak 1579, 1310, 1148, 1020, 850, 776, 612, and 499 cm−1 and the strong 1288 and 996 cm−1 spectral features (see Table 2 for bands assignment) point out the interaction between Pip7 and the colloidal Au surface. The broadness and pronounced intensity of the last two SERS bands additionally suggest the participation of the free electron-pair on the Pip7 nitrogen atom in this interaction. This is possible when the Pip7 ring adopts the tilted orientation. Also, Thi and Arg contribute to the SERS spectrum of [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK onto the Au surface. This conclusion is based on the appearance of the 1521, 1500[δ(CH)+ν(C=C), in-plane], 1365 [(δ(CH)+ν(C=C), in-plane], 1083 [ρ
r(CH), in-plane], 850 [δ(ring) + ν
(
CC
)
ring, in-plane], and 678 cm−1[(ν(CH), out-of-plane] bands due to Thi [31, 32]. The similar medium relative enhancement of the in-plane and out-of-plane Thi modes implies the tilted orientation of the Thi ring with respect to the Au nanoparticles surface. On the other hand, the interaction of the guanidine group with colloidal Au surface is manifested by the 1631, 1425, and 1365 cm−1 SERS signals.Addition of Aaa at theN-terminal end of [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK produces marked changes in the SERS spectral pattern as is evident in Figure 1(c) (the Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK analog). In the spectrum of Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK in the Au solution, the 1596, 1551, 1379, 1281, 1242, 1179, 1142, 1019, 881, 833, 652, and 464 cm−1 (see Table 2 for bands assignment) signals are due to Pip7. Among these, the 1379 and 1019 cm−1 bands exhibit the predominant relative intensity (Figure 1(c)) what point out on the vertical orientation of Pip7 on the Au surface. Although it seems that the Pip7 nitrogen atom does not participate directly with this surface (weak 1281 cm−1 band). Also, bands due to the Thi vibrations (1495, 1215, 1179, 1070, 881, 833, 671, and 568 cm−1) are weakly enhanced in the Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK SERS spectrum. This is why we suggest that Thi assists in the adsorption process onto the Au surface.In the SERS spectrum of [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK (Figure 1(d)), the phenyl ring (D-Phe7) modes (1608, 1026, and 1001 cm−1) are weakly enhanced. Unlike those, the bands mainly connected with the Thi5 ring dominate this spectrum (at 1504, 1360, 1165, 1093, 887, 840, and 682 cm−1). These results are in contrast to those obtained for the abovementioned analog adsorbed on the colloidal Ag surface [18] and suggest that the D-Phe7 ring is removed from the Au surface (no shift in the Phe bands wavenumber and width), whereas the Thi5 adopts the vertical arrangement on this surface. The appearance bands at 1284, 1251, 1151, 887, 840, 763, 729, and 594 cm−1 indicate that also Pip8 contributed to the SERS spectrum on Au surface. Of these bands, 1284 and 1151 cm−1 SERS signals (mainly enhanced) are due to the in-plane Pip8 ring vibrations. For this reason we imply that Pip8 assists in adsorption process and is oriented vertically on this substrate through the lone electron pair of the nitrogen atom. The Arg⋯Au interactions cannot be excluded for this peptide because of the 1639, 1608, 1438, 1165, 939, and 840 cm−1 spectral features. Based on these observations mainly the guanidine group appear to be in proximity to the Au surface (Table 2).In the case of Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK SERS spectrum (Figure 1(e)), both the Pip8 and Thi5 rings modes are enhanced. Pip8 gives rise to the 1570, 1275, 1181, 1144, 1013, 839, and 752 cm−1 spectral features, whereas the 1501 (the most intense, in-plane mode), 1377, 1144, and 670 cm−1 SERS signals are related to the Thi5 vibrations. Analysis of the Pip8 bands’ relative intensity, bands width, and bands wavenumber changes suggested that, similarly as in the case of [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Pip8 in Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK being tilted with respect to the colloidal Au surface interacts with this surface through the nitrogen-free electrons pair. However, it seems that the strength of this interaction is higher for Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK than for [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK. Much of the same analysis for the Thi5 bands implies that the Thi5 ring assists in the adsorption process adopting the near-edge-on orientation onto the colloidal Au surface. What is interesting in case of the Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK SERS spectrum (Figure 1(e)) is that we observed apart from bands at 1438, 1083, 936, 839, and 670 cm−1 very significantly enhanced overlapped, broad bands at 1602 [(δ(NH2
+) + ν
s(C=N)] and 1570 [δ(NH]. Based on these observations we suggest that we above all observed interaction between Arg’s –NH2-group from the guanidine group and Au surface.


## 4. Conclusions

In this study, we reported the mode of adsorption of BK and its potent B2 receptor antagonists: [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK adsorbed on the colloidal Au surface. We showed the following.(i)
The Phe5/Phe8 residues (in tilted orientation of the rings) are mainly involved in the BK interaction with the colloidal Au surface, whereas D-Phe7 in [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK is moved out of this surface; thus it is located on the opposite side of the polypeptide backbone.
(ii)
Pip and Thi adsorb on the colloidal Au surface for all the investigated BK analogues. The Thi and Pip residues in [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK adsorb in the tilted orientation on the Au surface (Pip through the piperidine nitrogen atom). Elongation of the peptide backbone by the addition of Aaa at the N-termini (the Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK analog) produces rearrangement of the Pip ring (it rises and there is no longer interaction between the piperidine nitrogen atom and Au) with respect to the Au surface and weakening of the Thi⋯Au interaction. On the other hand, the substitution of Pip7 by D-Phe7and Thi8 by Pip8(the [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK analog) triggers rise of Thi5 and Pip8 interacts mainly through the piperidine nitrogen atom and adopts vertical orientation with respect to the Au surface. Subsequent modification (the Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK analog) moves the Thi5 away from the Au surface and forces tilting of the Pip8 ring on this surface.
(iii)
The Arg residue of BK and its three analogues: [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK evidently participate in the adsorption process on the colloidal Au surface; the lack of Aaa SERS bands and the enhancement of the modes due to the residues from the C-terminal peptide end (at 5, 7, and 8 positions in the amino acid sequence) designate Arg at 9 position to be that one which interacts with the Au surface.



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*Source: 619373-2014-02-04.xml*