Tryptophan Tight Binding to Gold Nanoparticles Induces Drastic

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C: Plasmonics; Optical, Magnetic, and Hybrid Materials

Tryptophan Tight Binding to Gold Nanoparticles Induces Drastic Changes in Indole Ring Raman Markers Belen Hernandez, Lorenzo Tinacci, Yves-Marie Coïc, Alexandre Chenal, Régis Cohen, Santiago Sanchez-Cortes, and Mahmoud Ghomi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b02261 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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The Journal of Physical Chemistry

Tryptophan Tight Binding to Gold Nanoparticles Induces Drastic Changes in Indole Ring Raman Markers

Belén Hernández,a,b Lorenzo Tinacci,c Yves-Marie Coïc,d Alexandre Chenal,e Régis Cohen,f Santiago Sanchez-Cortes,c Mahmoud Ghomi*a,b

a

Laboratoire Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), UMR 7369, Université de Reims,

Faculté des Sciences, Moulin de la Housse, 51687 Reims cedex 2, France b

Sorbonne Paris Cité, Université Paris 13, Groupe de Biophysique Moléculaire, UFR Santé-Médecine-Biologie

Humaine, 74 Rue Marcel Cachin, 93017 Bobigny cedex, France c

Instituto de Estructura de la Materia, IEM-CSIC, Serrano 121, 28006-Madrid, Spain

d

Institut Pasteur, Unité de Chimie des Biomolécules, UMR 3523, 28 Rue du Docteur Roux, 75724 Paris cedex

15, France e

Institut Pasteur, Unité Biochimie des Interactions Macromoléculaires, UMR CNRS 3528, 25 Rue du Docteur

Roux, 75724 Paris cedex 15, France f

Service d’Endocrinologie, Centre Hospitalier de Saint-Denis, 2 Rue du Docteur Delafontaine, 93200

Saint-Denis, France

*Corresponding author: M. Ghomi, Tel: +33-1-48388928, E-mail: [email protected]

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ABSTRACT Experimental conditions favoring the tryptophan tight binding to the surface of citratereduced gold naoparticles (Cit-AuNPs), were explored. For this, the adsorption of three molecular compounds, free amino acid (Trp), tripeptide NH2-Gly-Trp-Gly-CONH2 and lanreotide, a synthetic cyclic somatostatin analogue, on large size (~70 nm) Cit-AuNPs, were analyzed. UV-visible absorption, transmission electron microscopy and surface-enhanced Raman scattering (SERS), were jointly used in the present investigation. At low pH (~3.5), when the repulsive electrostatic interactions between gold particles are sufficiently reduced, both peptides can induce strong NPs aggregation, leading particularly to substantial changes in the characteristic tryptophan Raman markers, as well as to the appearance of strong SERS markers at 1228 and 1113 cm-1. In contrast, upon increasing pH toward neutral value, the mentioned Trp Raman markers tend to adopt a spectral shape comparable to that observed in solution (bulk). In free amino acid, SERS effect could not be observed within a large pH interval, except at low pH upon increasing ionic strength. The reported data would be of benefit for following the adsorption processes that should be further considered in preparing Au-peptide nanodevices for therapeutic applications.


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INTRODUCTION Because of their unique optical properties, plasmonic nanostructures have gained an increasing interest in material science. Typically made of Au, Ag and Cu, i.e. metals with high reflectivity, plasmonic nanoparticles (NPs) exhibit strong UV-visible extinction bands, not present in bulk. In fact, the electrons at the metal surface, referred to as surface plasmon (SP), collectively oscillate at the same frequency as that of the incident electromagnetic wave, and subsequently radiate at the same frequency. When the incident wavelength (λ) falls nearby the principal plasmon absorption (λp), inducing the so-called surface plasmon resonance (SPR), the particles can absorb and scatter light out of their physical cross sections.1-2 This explains the brilliant color of the solution samples containing plasmonic colloids with a size much smaller than the excitation wavelength. SP oscillations may also decay through a non-radiative pathway to give rise to the absorption of light and its conversion to heat.3,4 The calorific capacity of plasmonic NPs, increasing with their size, is used in the applications requiring localized temperature elevation.4 The use of AuNPs in nanobiotechnology and nanomedicine is changing the traditional protocols, not only in molecular biology,5-7 but also in cell and tissue imaging, biodiagnostics, drug delivery and photothermal therapy.8-14 The reduced toxicity of AuNPs and their facile bioconjugation to antibodies and therapeutic agents through appropriately chemical groups (thiol, amine, sulfide, etc), permit selective targeting of cells and diseased tissues, thus motivating their use in various biomedical assays.3,4 The spectral features of AuNPs are found to be size- and shape-dependent. For instance, while gold nanospheres show their SPR extinction in the visible region, other nanoshapes such as nanoshells, nanorods, nanocubes and nanotriangles, are able to extend it over near-infrared to mid-infrared ranges, compatible with their application to biomedical purposes.15



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Synthesis of gold particles needs at least one reducing agent, ensuring the ionic→atomic gold conversion, as well as a NP stabilizer capping the formed particles, selected among small organic molecules, amino acids, polymers, and even large size biomacromolecules.16-22 Hereby, to modulate the electrostatic features of AuNPs, our choice was oriented toward trisodium citrate, used both as reductant and stabilizer. First initiated by Turkevich,16 the preparation of citrate-reduced gold nanoparticles (Cit-AuNPs) was further improved by Frens,17 allowing the particles with a size between 15 and 150 nm, to be synthesized. The main advantage of using citrate ions is due to the possibility of monitoring the protonationdeprotonation of their three carboxyl groups (pKa1=3.13, pKa2=4.76 and pKa3=6.40). As a consequence, around physiological pH, where the three citrate ion carboxyl groups are mostly deprotonated, large repulsive forces appear between gold particles covered by citrate ions, making difficult their close contacts and subsequent aggregation. In contrast, in a strongly acidic solution, carboxyl protonation favors NPs aggregation by reducing their repulsive interactions. This effect leads to the formation of the so-called hot spots, i.e. narrow interparticle junctions producing a huge amplification of both incident and scattered beams by NPs, from which SERS takes particularly benefit.23-25 Our main objective was to reveal the perturbations induced in Trp Raman markers upon binding onto AuNPs. For this, three molecular species were selected: (i) free AA (Trp) (Figure 1A); (ii) Cter-amidated tripeptide, NH2-Gly-Trp-Gly-CONH2 (Figure 1B); and finally (iii) a synthetic analogue of the natural hormone somatostatin26, referred to as lanreotide (Figure 1C), playing a pivotal therapeutic role for controlling hormone related symptoms of functioning neuro-endocrine-tumors and tumor growth.27-29 The cyclic structure of lanreotide is maintained by a disulfide linkage between its Cys2 and Cys7 residues, and the presence of the two adjacent D-Trp4-Lys5 central residues is considered as a key element in its interaction with somatostatin receptors.30-32


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MATERIAL AND METHODS Molecular compounds. Lyophilized sample of lanreotide/acetate salt was courtesy of IPSEN (Courtabœuf, France). Trp was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The tripeptide NH2-Gly-Tyr-Gly-CONH2 (hereafter referred to as GWG), was synthesized at the Institut Pasteur (Paris) according to the Fmoc/tBu solid-phase strategy33 on an ABI 433 synthesizer (Applied Biosystems, Foster City, CA). The details concerning the chemical synthesis were given in a previous paper.34 The purity (99.5%) of GWG was checked by RP-HPLC, applying a linear gradient of acetonitrile in aqueous TFA over 20 min at a 0.35mL/min flow rate on an Aeris Peptide 3.6 µm XB-C18 analytical (2.1 Å, 100 mm) column (Phenomenex, Le Pecq, France). The observed mass of GWG (340.1175 Da) was in accordance with the [M+Na+] expected mass (340.1386 Da). For lanreotide, the experimental data were consistent with its expected mass, i.e. monoisotopic [M+H]+ 1096.4743 d, observed 1096.4716 d. The net peptide content of GWG and lanreotide were determined by quantitative amino acid analysis.

Solution samples. Trp and peptides were dissolved in pure water taken from a Millipore filtration system (Guyancourt, France). Stock solutions were first prepared, as follows: Trp, 50 mM (10.2 mg/mL); GWG, 10 mM (3.19 mg/mL); lanreotide, 10 mM (10.96 mg/mL). They served to record bulk (solution) Raman spectra. Further dilution of these samples allowed reaching the concentrations needed for UV-visible absorption and SERS measurements. In certain experiments, to increase the ionic strength of a sample, sodium chloride, purchased from Merck (Fontenay-sous-Bois, France), and prepared at 150 mM concentration, i.e. 9 mg/mL), was added. Upon dissolution, the pH of samples was between



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5.5 and 6. Bulk Raman spectra of these compounds were measured without further pH adjustment.

Gold nanoparticles. HAuCl4 and citrate.Na3 (Merck) was used to prepare lots of of relatively large size (~70 nm) AuNPs, following the Frens protocol,17 i.e. upon addition of 0.30 mL of citrate solution (1% by weight) to the boiling solution of HAuCl4 (10-2 % by weight).25 The pH value of colloidal solutions measured just after elaboration was ~3.5, adjusted to higher values by adding drops of NaOH (1N) (Merck).

Experimental setups. UV-visible spectra of silver colloids, either alone or in the presence of peptides, were recorded at room temperature on a Shimadzu 3600 UV-visible absorption spectrometer (Duisburg, Germany) by means of quartz cells having an optical path of 1 cm. Transmission electron microscopy (TEM) images were taken at room temperature using a JEOL JEM-2010 electron microscope (Croissy, France) with an acceleration voltage of 200 kV. The samples were prepared by depositing 10 µL of the suspension containing either AuNPs, or their complexes with peptides, on carbon coated Cu grids (ref. G400-Cu). Room temperature Stokes Raman spectra were analyzed in solution samples at right angle on a Jobin-Yvon T64000 spectrometer (Longjumeau, France) at single spectrograph configuration, 1200 grooves/mm holographic grating and a holographic notch filter. Raman data corresponding to 1200 s acquisition time for each spectrum were collected on a liquid nitrogen cooled CCD detection system (Spectrum One, Jobin-Yvon). The effective slit width was set to 5 cm-1. Solution samples were excited by the 488 nm line of an Ar+ laser, Spectra Physics (Evry, France), with 200 mW power at the sample. SERS data were collected on an InVia spectrometer (Renishaw Ibérica S.A., Gavá, Spain) equipped with an electrically cooled CCD camera. Excitation wavelength was emitted from a 785 nm diode laser, working at 50% of its power, i.e. 150 mW. As ~90% of the emitted power is dispelled by different optical devices, redirecting the laser beam to the sample, the average


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power at the sample was ~18 mW. MacroRaman objective was used to focus the laser beam on the samples. Spectra were analyzed by a 1200 grooves per mm holographic grating. The spectral resolution was 2 cm-1, with a total acquisition time of 10 seconds per SERS measurement. The absence of intense and broad SERS bands assignable to burned organic samples between 1250 and 1600 cm-1 proved the integrity of the analyzed molecules. Moreover, no interference can be expected between the SERS spectra from amino acid/peptides and citrate ions covering the surface of AuNPs. We have recently reported the SERS data obtained from the covering citrate ions, as well as their degradation products formed as a function of time.25

Post-record spectroscopic data treatment. Buffer subtraction and smoothing of the observed spectra was performed using the GRAMS/AI Z.00 package (Thermo Galactic, Waltham, MA, USA). Final presentation of Raman spectra was done by means of SigmaPlot package 6.10 (SPSS Inc., Chicago, IL, USA).

Quantum mechanical calculations. Energetic and geometrical data of Trp, GWG and lanreotide were estimated by the density functional theory (DFT) approach,35 using the hybrid B3LYP functional.36,37 Polarized triple zeta basis sets, referred to as 6-311++G(d,p), were used for Trp and GWG. Taking into account the structural complexity of lanreotide (composed of 8 amino acids, and 148 atoms), the computational time was decreased by using a less extended basis set, i.e. polarized double zeta 6-31+G(d). The hydration effect was considered by a purely implicit model, which consists in placing the solute in a polarizable continuum medium (PCM),38 of which the relative permittivity was supposed to be that of water (εr=78.39). Harmonic vibrational calculations were carried out to make sure that an optimized geometry can be accepted as that corresponding to a local minimum through the absence of any imaginary frequency. All quantum mechanical calculations were made using the Gaussian09 package.39 Base superposition error (BSSE) was not corrected, because in a



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recent work,40 it was shown that this correction has a negligible effect on the relative energy order of the calculated conformers.

RESULTS AND DISCUSSION pH Range, Molecular Charge and Particle Size. All the reported experimental data were obtained within the 3.5-to-7 pH interval. In this range (i) Trp, having two pKa values (namely pKa1=2.38 and pKa2= 9.39) corresponding to its carboxyl and amine groups, respectively, is naturally in a zwitterionic (NH3+/COO-) form (Figure 1A); (ii) GWG remains cationic with only one positive charge (NH3+) at its Nter group (Figure 1B); and (iii) lanreotide also keeps a cationic character, with two positive charges borne by the two amine groups located at Nter and at Lys5 side chain head group (Figure 1C). Moreover, the pKa associated to the deprotonation of a Lys side chain is above 10, and the indole ring cannot be deprotonated in the considered pH range. It has been shown recently that large size Cit-AuNPs (typically ≥40 nm) can be used as optimal SERS substrates for cationic peptides.25 Here, AuNPs having an average size of ~70 nm with a rather ellipsoidal shape (Figure 2A), were used. Electrostatic and plasmonic stability of these particles was recently followed over a long period (90 days).25 Particularly, the ζ-potential values of these AuNPs, showing a limited variation (ζ=-36±3 mV) were consistent with their negatively charged surfaces.25

Extinction Profiles of Gold Particles. AuNPs manifest a single broad extinction band peaking at 539 nm (Figure 3A). At pH~3.5, at most one of the citrate carboxyl groups is deprotonated. When pH is increased toward the neutral value, an overall increase of the extinction band is observed (Figure 3A). However, upon increasing ionic strength, a notable decrease of the plasmonic extiction appears, accompanied by the appearance of a broad band centered at ~750 nm (Figure 3A). This effect is generally assigned to the aggregation of 8 ACS Paragon Plus Environment

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plasmonic particles, brought about by the presence of positively charged counterions (Na+), allowing the negative citrate ions, to be screened.25

Gold Particles Aggregation Induced by Cationic Peptides. As evidenced by the plasmonic band redshift, the adsorption of GWG and lanreotide on negatively charged plasmonic surfaces also lead to NPs aggregation (Figure 3B and C). Taking into account weak inter-particle repulsive forces at pH ~3.5, a lower concentration of GWG (10-6 M) was required to induce NPs aggregation (Figure 3B). Close to neutral pH, upon increasing ionic strength, a higher peptide concentration (10-5 M) was necessary to achieve the same objective. Similar behavior was observed in lanreotide adsorbed on AuNPs (Figure 3C). As mentioned in Introduction section, SERS activation basically depends on the aggregation of colloidal particles. The selected laser wavelength (λL=785 nm) falls perfectly within the redshifted plasmonic extinction bands, ensuring the enhancement of the Raman signal by adsorbed peptides (Figures 3B and C).

SERS Markers of Free amino Acid. It is to be mentioned that bulk Raman spectrum of Trp (Figure 4A) were fully assigned previously.41 Here, we limit our discussion to a selection of eight characteristic Trp Raman markers (referred to as W1-to-W8). Observed in the middle wavenumber region (1650-700 cm-1), all these markers originate from the indole ring vibrational modes.41 (Table 1). Figure 4B and C display the SERS data of Trp obtained at pH ~3.5 in the presence of 150 mM NaCl and 20 mM KNO3, respectively. The similarity of these spectra proves that the SERS effect is independent of the counterion type (Na+ vs K+, or Cl- vs NO3-) used to reinforce NPs aggregation. It is noteworthy that all our attempts to records SERS data from Trp at higher pH values (pH>6) were unsuccessful. Even at low pH (~3.5), where NPs aggregation is generally facilitated, SERS effect could only be obtained by increasing ionic



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strength. This means that to favor NPs aggregation, addition of positively charged counterions (Na+ or K+) is required for screening the terminal COO- of the zwitterionic Trp (Figure 1A). SERS data of Trp (Figure 4B and C) reveal drastic changes in the intensities and wavenumbers of Trp markers (Figure 4A). Starting with the most striking effect, the presence of two strong SERS markers at ~1230 and ~1115 cm-1 (hereafter referred to as Ws1 and Ws2, respectively, Table 2), should be mentioned. Other important changes are also to be noticed: (i) W1, W2 and W3 modes observed in bulk at 1621, 1579 and 1552 cm-1, respectively (Figure 4A) are collectively downshifted by ~25-to-28 cm-1 in SERS (Figure 4B); (ii) the doublet W4/W5 located at 1365/1343 cm-1 in bulk (Figure 4A) merges into one strong and broad band peaking at 1359 cm-1 in SERS (Figure 4B); (iii) the most intense Trp band (W6) observed at 1012 cm-1 (Figure 4A) is downshifted by 16 cm-1, and observed at 996 cm-1 in SERS (Figure 4B); (iv) the last two markers, W7 and W8, observed in bulk at 880 and 758 cm-1, respectively (Figure 4A), present comparatively moderate downshifts, i.e. to 871 and 756 cm-1, respectively (Figure 4B). It should be emphasized that the DFT calculations performed on Trp complexes with large size Au assemblies, have previously predicted possible interaction of indole ring with a gold surface.42

SERS Markers of Cationic Peptides. In contrast to the SERS data observed in Trp, those recorded from GWG and lanreotide could be analyzed within a large pH range and as a function of ionic strength. These spectra observed in the whole middle wavenumber region (1750-500 cm-1) are displayed in Figures S1 and S2 (Supplementary Information). In the main text, keeping in mind the most striking effects observed in the Trp SERS spectra (vide supra), we undertake our discussion on cationic peptides through three characteristic spectral regions, as follows: - The first region (1300-1050 cm-1) (Figure 5) shows the evolution of the two SERS markers (Ws1 and Ws2, Table 2). At pH~3.5, the GWG SERS data show these markers at 1228 and


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1113 cm-1 (Figure 5B bottom). Upon increasing pH and ionic strength, the intensity of both SERS markers decreases, while their wavenumbers are upshifted (Figure 5C and D bottom). Similar effects are observed in lanreotide (Figure 5B-D top, Table 2). In lanreotide two characteristic Tyr3 modes at 1208 and 1175 cm-1, referred to as Y3/Y4,34 are also observed in the same spectral region. No considerable wavenumber shift has been detected for these modes in going from bulk to SERS data (Figure 5A-D top). - The second spectral region (1750-1300 cm-1) (Figure 6) contains five out of the eight Trp characteristic modes, i.e. W1-to-W5 (Table 1). At pH~3.5, due to a considerable enhancement in amide I region (1700-1600 cm-1), it becomes difficult to follow the behavior of W1 in the SERS data of lanreotide (Figure 6B top). The strongest Raman marker in lanreotide, arising from D-Nal1 residue,43 is observed at 1384 cm-1 (Figure 6A-D top). This mode is neither altered by pH, nor by ionic strength. At higher pH and ionic strength (Figure 6C and D), the wavenumbers corresponding to W1, W2 and W3 modes are progressively upshifted toward the values corresponding to bulk Raman data (Figure 6A). The strong single band (at ~1355 cm-1) observed at low pH (Figure 6B) appears as a resolved doublet (W4/W5) (Figure 6C and D), as observed in bulk (Figure 6A). - The third region (1050-700 cm-1)(Figure 7) contains the three markers W6, W7 and W8 (Table 1). In lanreotide, the presence of two D-Nal1 markers,43 at 1017 cm-1 (close to W6) and 770 cm-1 (close to W8), should also be noted. Both D-Nal1 markers remain unaffected in SERS spectra. Note also the presence of a well known Tyr3 doublet, referred to as Y5/Y6,34 observed at 852/828 cm-1 in bulk (Figure 7A top), remaining unaffected by the adsorption of lanreotide on AuNPs. It is worth noting that at pH~3.5, an important wavenumber downshift (1012→996 cm-1) is observed for W6 marker (Table 1). Upon increasing pH toward the neutral value, W6 is first upshifted to ~1000/1012 cm-1 (pH~6, Figure 7C), before moving back to its initial wavenumber at 1012 cm-1 (pH~7, Figure 7D). Moderate downshift



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(880→872 cm-1) is observed for W7 (Figure 7B), getting gradually its initial wavenumber at higher pH (Figure 7C and D). As in free amino acid, W8 does not show a considerable wavenumber shift from bulk to SERS data (Figure 7A-D).

Comparison with Previously Reported SERS Data. Thanks to a strong modulation of the electronic polarizability, the vibrational modes of aromatic compounds give generally rise to intense Raman bands.34,43-44 This was presumably the reason why the first published SERS data were collected from pyridine adsorbed on silver electrode.45 A decade later, using Ag colloids the first SERS data of aromatic amino acids,46 as well as those from short peptides and soluble proteins, were reported.46-49 Recently, AgNPs were used to compare the data from aromatic amino acids by traditional SERS (excitation at 532 nm) and surfaceenhanced hyper-Raman scattering (SEHRS) (double-photon excitation at 1064 nm).50 From all the reported SERS investigations, it can be deduced that the adsorption on AgNPs does not notably affect aromatic wavenumbers.51-53 In other words, aromatic amino acids do not enter in tight interaction with Ag surface.51-53 This has been corroborated by the DFT calculations on Trp-Ag complexes, showing that the zwitterionic amino acid preferentially binds to silver via its terminal charged groups.54 In contrast, the presently reported SERS data on AuNPs revealed substantial changes in Trp markers (Tables 1 and 2). However, all these changes mainly appear at low pH. In a recent work by daFonseca et al.,55 the SERS data obtained from Trp and Trp-containing short peptides adsorbed on smaller size Cit-AuNPs (apparently ~30 nm regarding the published extinction spectra), were consistent with wavenumber changes in certain indole ring markers upon addition of HCl (amount not specified). A counter example concerns the SERS data collected from the free amino acid adsorbed on gold nanoshells (Au/SiO2), in which no substantial wavenumber shift was announced for Trp markers.44


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At last, we should emphasize a set of the previously reported SERS data from Trpcontaining peptides on metal electrodes (Ag, Au and Cu).56 Although the spectral shape of the SERS data was shown to be electrode type-dependent, very interesting points appear upon comparison the SERS data collected from Au electrode with those corresponding to Au colloids (present data). In particular, substantial changes in Trp markers should be pointed out upon variation of Au electrode potential from -1.2 V to +2 V. Among them, the strong band appearing at 1358 cm-1 (replacing the well known W4/W5 Trp doublet at ~1360-1340 cm-1), along with the enhancement of the two bands at 1239 and 1117 cm-1 (which can be correlated to Ws1 and Ws2 SERS markers). From this comparison, it can be concluded that the data obtained on a positive potential Au electrode can be assimilated to those collected at low pH, i.e. upon lowering repulsive interactions between Au colloids. In opposite, the data obtained at the ultimate negative potential (-1.2 V) can be related to those collected at pH 7, i.e. when high repulsive interactions exist between negatively charged Cit-AuNPs.

Low Energy Conformers of Molecular Compounds. To suggest reliable binding schemes for explaining the adsorption of the molecular compounds on AuNPs (see last part of this section), it is first necessary to search for the representative low energy conformers. Recent theoretical analysis on all aromatic AAs,40 have mentioned that the global conformation of a free Trp is mainly governed by two torsion angles, named χ1 and χ2, defined around the side chain Cα-Cβ and Cβ-Cγ bonds, respectively (Figure 1A). Through the generation of the most plausible (χ1,χ2) rotamers, the lowest energy conformer of a zwitterionic Trp embedded in a solvent continuum was assigned to its side chain g-g+ orientation (where g-: gauche- and g+: gauche+), corresponding to the values (χ1=-56.9°, χ2=+107.5°). For graphical representation of this conformer see Figure 1A. The tripeptide GWG can obviously generate a large number of possible rotamers regarding both the Trp side chain (χ1,χ2), and the backbone (ϕ, ψ and ω) conformational angles. The 13 ACS Paragon Plus Environment


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details on the GWG low energy conformers is out of the scope of this report and will be mentioned in a forthcoming paper. Briefly, the g-g+ orientation of the Trp side chain (χ1=64.4°, χ2=+100.2°) remains the most energetically favorable one, while the peptide backbone prefers an extended conformation at both ends (i.e. in Gly1 and Gly3 residues), and a β-strand like in its middle (Trp2 residue) (Table 3). For graphical representation, see Figure 1B. Atomic Cartesian coordinates of this conformer are reported in Table S1 (Supplementary Information). Based on a former investigation on low concentration structural features of lantretide by means of UV-circular dichroism and Raman spectroscopy, a stable type-II’ β-turn was suggested for this octapeptide.43 However, no geometrical data was unfortunately available for lanreotide. To suggest an acceptable low energy structure, we used as the starting point a similarly structured somatostatin analogue, i.e. a cyclic octapeptide referred to as octreotide, also adopting a type-II’ β-turn around its central D-Trp4-Lys5 residues.53 Replacing the amino acids differing lanreotide from octreotide (i.e. those located at the first, third, sixth and eighth positions), the whole structure was geometry optimized. The graphical representation of the optimized conformer is displayed in Figure 1C. See Table 3 for backbone torsion angles, and Table S2 (Supplementary Information) for atomic Cartesian coordinates. It is worth mentioning that the Trp side chain orientation in lanreotide is predicted to be tg+ (where t: trans), corresponding to the values (χ1=+179.1°, χ2=+107.6°). The g-→t reorientation of χ1 in lanreotide (with respect to Trp and GWG) is presumably due to the favorable aromaticaliphatic interactions occurring between the side chains of the two adjacent

D-Trp

4

and Lys5

residues (Figure 1C).

Origin of Trp SERS Markers (Ws1 and Ws2). In this framework, we adopted a rationale assuming that the strong Ws1 and Ws2 markers (observed at 1230 and 1115 cm-1) (Figure 4B and C) should originate from two indole ring vibrational modes observed with 14 ACS Paragon Plus Environment

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weaker intensities in bulk Raman spectra (Figure 4A). Given this, the behavior of the two weak Raman bands observed in free amino acid at 1228 and 1133 cm-1 received particularly our attention. Our assumption has been reinforced by the fact that the SERS data collected from cationic peptides (Figure 5) also reveal two strong SERS markers at 1228 and 1113 cm-1 at low pH (Figure 5B), loosing their intensity upon increasing pH and ionic strength (Figure 5C and D). Particularly, at pH~7 (Figure 5D) one can notice two weak bands whose wavenumbers are close to those observed in bulk, i.e. 1235 and 1130 cm-1 (Figure 5A). To check the validity of the above mentioned assumption, we resorted to the vibrational calculations on the lowest energy conformer of GWG (Figure 1B) embedded in a solvent continuum. Among the calculated data, one can find two modes at 1230 and 1130 cm-1 (Figure 8A and B). Both modes are assigned to the coupling between the C-N bond-stretch and C-N-H angular bending in indole ring (Figure 8A and B bottom).

Possible Binding Schemes of Molecular Compounds. Recent investigations based on the analysis of the low wavenumber SERS markers have evidenced that the binding of cationic peptides to AgNPs is of electrostatic type, made possible through the attractive interaction between their Nter positively charged amino group (NH3+) and the negatively charged chloride (Cl-) ions covering silver surfaces.51-53 The analysis of the adsorption of amino acids19-22 and peptides52,53 on AuNPs were also consistent with the fact that their positively charged Nter group should be considered as the most appropriate anchoring sites on gold particles. As a consequence, upon adsorption the Trp side chain involved in free amino acid, as well as in GWG and lanreotide would be naturally oriented toward surrounding medium (Figure 9). Hence, at low pH, where the repulsive interactions between Cit-AuNPs are sufficiently low, the indole ring can suffer a tight binding with an approaching gold particle (Figure 9A-C), explaining the drastic changes observed in the present SERS data (Figures 4 and 5, Tables 1 and 2). It should be remarked that in lanreotide, a possible

D-Trp

4



Page 16 of 38

binding to AuNPs might be reinforced by the additional favorable interactions occurring between the adjacent Lys5 side chain head group (NH3+) and the negatively charged metal surface (Figure 9C). At higher pH values (for instance pH≥6), the increase in inter-particle repulsive interactions leads naturally to the loss of the mentioned tight interaction (Figure 9AC). This might be the reason why the characteristic SERS markers tend to get a spectral shape similar to that observed in bulk. Finally, the binding scheme for free amino acid, displayed in Figure 9A, allows us to better apprehend the reason why to activate SERS effect at low pH, the unfavorable repulsive interaction of the terminal COO- and an approaching AuNP should be necessarily screened by an additional counterion (Na+/K+) through the increase of ionic strength increasing in colloidal solution. At higher pH, even the presence of additional positive counterions cannot completely counterbalance the repulsive interactions between Cit-AuNPs, avoiding the formation of necessary hotspots for activating SERS effect in free Trp.

CONCLUDING REMARKS The simultaneous analysis of three molecular compounds with increasing structural complexity (amino acid→tripeptide→octapeptide), all three containing one indole ring in their structure, allowed us to probe the interaction of tryptophan with the surface of negatively charged Cit-AuNPs. SERS data derived from the selected molecular compounds were collected as a function of pH and solution ionic strength. It was clearly evidenced that at low pH, where the repulsive interactions between colloidal particles are sufficiently lowered, the appearance of two strong SERS markers (referred to as Ws1 and Ws2, see Table 2), as well as the substantial spectral changes observed in the traditional Trp Raman markers (W1-W8, see Table 1), can be used as “efficient indicators of Trp tight contact with plasmonic gold surfaces”.


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However, the ability of tryptophan, as the only aromatic amino acid that is able to produce a tight binding to plasmonic gold surfaces, is noteworthy. Other aromatic residues, such as the Nal and Tyr residues involved in lanreotide (Figure 1B), the Phe residues included in somatostatin and octreotide,52,53 or even larger size synthetic aromatic residues in pasireotide,53 were shown to be unable to induce similar spectral changes. In contrast, when the peptide chains is adsorbed on AgNPs, tryptophan behaves similarly to other aromatic residues, i.e. without any remarkable wavenumber change of its characteristic Raman markers.51-53 Around physiological pH, the Trp tight binding disappears, presumably because of the strong repulsive interactions of Cit-AuNPs. In this situation, the preferential binding site of a free Trp, as well as GWG and lanreotide, remains their common Nter positively charged amino group (NH3+). This allows their unique indole ring to be oriented toward the surrounding medium, possibly used as a functional group creating favorable interactions with other molecular partners. Many previously reported examples, based on the functionalized gold particles with Trp, can justify this assumption. For instance, (i) Trp-functionalized AuNPs are used for deep UV Imaging of microbial cells;57 (ii) Trp-coated AuNPs were shown to inhibit amyloid aggregation of insulin;58 (iii) Trp-capped bimetallic gold/silver NPs were shown to present a reduced hepato- and nephro-toxicity during in vivo applications.20 Furthermore, it has also been shown that Trp can be used as reductant and stabilizer of AuNPs between 20 and 30 nm.22,59 During the recent years, because of their better addressability, the functionalized plasmonic nanoparticles with somatostatin analogues have received much attention in clinical therapy. It should be recalled that certain somatostatin receptors are overexpressed in the primary tumour or its metastases.60 Preliminary tests have already highlighted the cellular uptake of somatostatin-coated AuNPs on different tumour cell lines.11 The temperature



Page 18 of 38

increase induced by irradiation during 5 ns at 532 nm, falling within the extinction band of octreotide-coated AuNPs, leads to a notable decrease of HELA cells viability.12 Furthermore, somatostatin receptor-based imaging was shown to be efficient for staging, therapy planning, and follow-up. In particular, when coupled to radiolabeled somatostatin analogues, this imaging acquires high specificity, low antigenicity, rapid clearance, and good tissue distribution.13,61,62 As a consequence, the future synthesis of multifunctional nanodevices coated with radiolabeled somatostatine analogues, appears to be extremely promising in tumour diagnosis and therapy. In this framework, the presented data would help to follow the Au-peptide adsorption mechanisms on gold nanostructures.

Supporting Information Figures S1 and S2, Tables S1 and S2, as well as the full reference 39, have been provided. Figure S1. Raman and SERS data of the tripeptide GWG in the whole middle wavenumber region. Figure S2. Raman and SERS data of the octapeptide lanreotide in the whole middle wavenumber region. Table S1. Cartesian coordinates of the lowest energy conformer of GWG. Table S2. Cartesian coordinates of the optimized conformer of lanreotide. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS The authors would like to thank IPSEN (Courtabœuf, France) to have generously provided the lanreotide samples used in this work. The theoretical calculations described here were granted access to the HPC resources of CINES/IDRIS under the allocations A0010805065 and A0030805065 made by GENCI (Grand Equipement National de Calcul Intensif) during


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the two years 2017-2018. This work was supported by Spanish Ministerio de Economía, Industria y Competitividad (projects FIS2014-52212-R and FIS2017-84318-R).

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Table 1. Characteristic vibrational modes of tryptophan observed in bulk and on gold nanoparticles.a Trp

Trp@bulk (10-2 M)b

W1 W2 W3 W4 W5 W6 W7 W8

1621 1579 1552 1365 1343 1012 880 758

GWG

GWG@bulk (10-2 M)e

W1 W2 W3 W4 W5 W6 W7 W8

1620 1578 1552 1360 1340 1012 880 760

Lanreotide

Lanreotide@bulk (10-2 M)k

W1 W2 W3 W4 W5 W6 W7 W8

1617 1579 1552 1360 1340 (sh) 1012 880 760 (sh)

Trp@Au (10-5 M) pH~3.5/150 mM NaClc 1594 1554 1524 1355* (W4/W5)

Trp@Au (10-5 M) pH~3.5/20 mM KNO3d 1596 1556 1525 1359* (W4/W5)

996 871 756

996 873 756

GWG@Au (10-6 M) pH~3.5f 1593 1555 1521 1354* (W4/W5) 996 874 (sh) 755 (sh)

GWG@Au (10-5 M) pH~6/150 mM NaClg 1601 1555 (sh) 1531 1360 1344 (sh) 1012/1002 (sh) 876 760

GWG@Au (10-5 M) pH~7/150 mM NaClh 1620 -----1540 1360 1344 1012 876 760

Lanreotide@Au (10-7 M) pH~3.5l

Lanreotide@Au (10-7 M) pH~6/150 mM NaClm

Lanreotide@Au (10-6 M) pH~7/150 mM NaCln

1565 1524 1356 (W4/W5)

1567 1531 1360 1343 (sh) 1000 (sh) 874 (sh) 760 (sh)

1619 1559 1534 1360 (sh) 1343 1013 874 760 (sh)

996 (sh) 874 (sh) 755 (sh)

a Observed wavenumbers are in cm-1. W1-to-W8 refer to the eight characteristic Raman markers of Trp. (sh) Raman bands observed as shoulders. * W4 and W5 modes merge together to give rise to a strong nonresolved band. In parentheses sample molar concentrations are reported. b See Figure 4A; cSee Figure 4B; dSee Figure 4C. e See Figure 6A bottom; fSee Figure 6B bottom; gSee Figure 6C bottom; hSee Figure 6D bottom. k See Figure 6A top; lSee Figure 6B top; mSee Figure 6C top; nSee Figure 6D top.


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Table 2. SERS markers of tryptophan.a

a

Trp

Trp@bulk (10-2 M)b

Ws1 Ws2

1228 (w) 1131 (w)

GWG

GWG@bulk (10-2 M)e

Ws1 Ws2

1237 (w) 1130 (w)

Lanreotide

Lanreotide@bulk (10-2 M)k

Ws1

1235 (w)

Ws2

1128 (w)

Trp@Au (10-5 M) pH~3.5/150 mM NaClc 1229 (s) 1115 (s)

Trp@Au (10-5 M) pH~3.5/20 mM KNO3d 1231 (s) 1116 (s)

GWG@Au (10-6 M) pH~3.5f 1228 (s) 1113 (s)

GWG@Au (10-5 M) pH~6/150 mM NaClg 1230 (m) 1118 (m)

GWG@Au (10-5 M) pH~7/150 mM NaClh 1232 (w) 1119 (w)

Lanreotide@Au (10-7 M) pH~3.5l 1228 (s)

Lanreotide@Au (10-6 M) pH~6/150 mM NaClm 1230 (m)

Lanreotide@Au (10-6 M) pH~7/150 mM NaCln 1232 (w)

1113 (s)

1118 (m)

1119 (w)

-1

Observed wavenumbers are in cm . Ws1 and Ws2 refer to the two characteristic SERS markers of Trp. (s) strong; (m) middle; (w) weak; (sh) shoulder. In parentheses sample molar concentrations are reported. b See Figure 4A; cSee Figure 4B; dSee Figure 4C. e See Figure 5A bottom; fSee Figure 5B bottom; gSee Figure 5C bottom; hSee Figure 5D bottom. k See Figure 5A top; lSee Figure 5B top; mSee Figure 5C top; nSee Figure 5D top.

25

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-130.7 +146.4 +176.0

Gly3

ϕ ψ ω

+179.3 +179.5 -----

-156.8 +115.1 +174.4

Tyr3

ϕ ψ ω

-143.1 +105.4 -173.6

D-Trp4

ϕ ψ ω

+57.4 -126.3 -179.8

Lys5

ϕ ψ ω

-78.9 -13.7 +178.3

Val6

ϕ ψ ω

-84.6 +82.6 -165.5

Cys7

ϕ ψ ω

-136.6 +47.5 +164.9

Thr8

ϕ ψ ω

+123.0 -58.9 -----

β-strand

ϕ ψ ω

Type-II’ β-turn

Cys2

Conformation

β-strand

ϕ ψ ω

Backbone Angles ----ϕ -179.7 ψ +174.1 ω

Extended

Trp2

Extended

Table 3. Backbone conformational angles of GWG and lanreotide.a GWG Lanreotide Residue Backbone Angles Conformation Residue Gly1 ----D-Nal1 ϕ -168.8 ψ -179.9 ω Extended β-strand

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

a

Conformational angles are expressed in degrees. Amino acid residues are numbered from Nter to Cter in both peptides (GWG and lanreotide) Conformational feature of each backbone segment is also reported. Cartesian coordinates of these optimized conformers are provides in Tables S1 (GWG) and S2 (lanreotide) (Supplementary Information).


Page 27 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions

Figure 1. Chemical composition of free amino acid, (A), tripeptide Gly-Trp-Gly (B) and lanreotide (C). These Figures correspond to the geometry optimized conformers by DFT calculations in a polarized continuum (see text for details). Hydrogen atoms are removed in lanreotide. Backbone torsion angles of GWG and lanreotide are reported in Table 3. For their atomic Cartesian coordinates, see Tables S1 and S2 (Supplementary Information).

Figure 2. Tranmission electron microscopy images obtained from the samples containing gold particles (A), GWG (10-6 M) (B), and lanreotide (10-7 M) (C) adsorbed on AuNPs.

Figure 3. Extinction spectra of the samples containing AuNPs (A), AuNPs in presence of GWG (B) lanreotide (C). pH, ionic strength and molecular concentration of each sample are reported.

Figure 4. Room temperature bulk Raman (A) and surface-enhanced Raman (B and C) data from free amino acid (Trp). pH and molar concentration are reported. NaCl and KNO3 mean that ionic strength is increased either by 150 mM sodium chloride or 20 mM potatium nitrate, respectively. Characteristic Trp Raman markers as designated by W1-to-W8 are indicated in red color. The blue frame contains the two Trp SERS markers (Ws1 and Ws2) as marked by asterisks.

Figure 5. Room temperature bulk Raman (A) and surface-enhanced Raman data (B-D) of GWG (bottom row) and lanreotide (top row) recorded in the 1300-1150 cm-1 spectral region. pH and molar concentration are reported. NaCl means that ionic strength is increased by 150 mM sodium chloride. The positions of the two Trp SERS markers, Ws1 and Ws2, are also indicated in red color.



Page 28 of 38

Figure 6. Room temperature bulk Raman (A) and surface-enhanced Raman data (B-D) of GWG (bottom row) and lanreotide (top row) recorded in the 1750-1300 cm-1 spectral region. pH and molar concentration are reported. NaCl means that ionic strength is increased by 150 mM sodium chloride.The positions of the five characteristic Trp markers, designated by W1to-W5, are indicated. Y3 and Y4 refer to tyrosine markers, and Nal indicates a naphtylalanine Raman marker. Figure 7. Room temperature bulk Raman (A) and surface-enhanced Raman data (B-D) of GWG (bottom row) and lanreotide (top row) recorded in the 1050-700 cm-1 spectral region. pH and molar concentration are reported. NaCl means that ionic strength is increased by 150 mM sodium chloride.The positions of the three characteristic Trp markers, designated by W6to-W8, are indicated. Y5 and Y6 refer to tyrosine markers, and Nal indicates a naphtylalanine Raman marker.

Figure 8. Graphical representation of the two calculated vibrational modes, supposed to be the precursors of the two Trp SERS markers (Ws1 and Ws2). Calculated modes are from the DFT calculation on the tripeptide Gly-Trp-Gly placed in a polarized solvent continuum (see text for details). The calculated wavenumber of each mode (scale factor=0.985), as well as its assignment in terms of internal coordinates are reported. For the sake of clarity the two extreme residues, i.e. Gly1 and Gly3 are only shown by their respective Cα atoms.

Figure 9. Suggested adsorption schemes for the three molecular compounds Trp (A), GWG (B) and lanreotide (C) on citrate-reduced gold nanoparticles. Molecular representations are made possible by means of the geometry optimized conformers displayed in Figure 1 (see text for details). Red and green arrows show the relative motion of the two interacting nanoparticles upon decreasing and increasing pH, respectively.


Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


A

B

C

+ D-Trp4

Trp2

Lys5 Tyr3

χ1

χ1 Nter

Cter

-

+

Val6

χ2

χ2

Cys7

Cys2 Cter

Nter

Nter

+ Gly

1

Cter

Thr8

+

Gly3 D-Nal1

Trp

GWG

Lanreotide Nal: naphtylalanine

Figure 1. Chemical composition of free amino acid, (A), tripeptide Gly-Trp-Gly (B) and lanreotide (C). These Figures correspond to the geometry optimized conformers by DFT calculations in a polarized continuum (see text for details). Hydrogen atoms are removed in lanreotide. Backbone torsion angles of GWG and lanreotide are reported in Table 3. For their atomic Cartesian coordinates, see Tables S1 and S2 (Supplementary Information). 29


The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A

B

Page 30 of 38

C Lanreotide@Au

AuNPs GWG@Au

200 nm

Figure 2. Tranmission electron microscopy images obtained from the samples containing gold particles (A), GWG (10-6 M) (B), and lanreotide (10-7 M) (C) adsorbed on AuNPs. 30


Page 31 of 38

A

B

C

AuNPs

GWG@Au

Lanreotide@Au

542

539

0,22

0,20 0.2

0,25

Lan 10-7 M / pH~3.5 -7 Lan 10 M / pH~6 / 150 mM NaCl -6 Lan 10 M / pH~7 / 150 mM NaCl

GWG 10-6 M/ pH~3.5 GWG 10-5 M / pH~6 / 150 mM NaCl GWG 10-5 M / pH~7 / 150 mM NaCl

0.18 0,18

544

pH~3.5 pH~6 pH~7 pH~6 / 150 mM NaCl pH~7 / 150 mM NaCl

0.3 0,30

785

0,16

785

0,18

0.2 0,20 0,16

785

Extinction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


0,15

0,14

0,12

0,14

0.10,10

0,12

0.1 0,10 0,10 0.1

0,08

0,08

0,05

0,06 0,06 0,00 0

0,04 400

500

600

700

800

400

500

600

700

800

400

500

600

700

800

Wavelength/nm

Figure 3. Extinction spectra of the samples containing AuNPs (A), AuNPs in presence of GWG (B) lanreotide (C). pH, ionic strength and molecular concentration of each sample are reported. 31



Trp SERS markers

W3 1525

Raman intensity

1359

W4/W5

WS1 WS2

*

996 1229

755 771

873 1012

861

871

936

1147

781

756

996

1115

1355 1471 1451 1421

1554 1552

10-5 M pH 3.5/NaCl

W8 758

W4

1400

1200

880

5x10-2 M

1000

710

862

970

1133 1110 1078

1259 1228 1205

1494

W@bulk

W7 1305

1460

1365 1343

1435

W2 W1

1600

W@Au

W6

W5

1800

10-5 M pH 3.5/KNO3

*

W3

A

W@Au

936

1148

1596

W8

*

1690

1594

B

W6

W7

1524

1680

W1

1556

C

1116

1231

*

1472 1452 1421

W2

1621 1579

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 32 of 38

800

600 -1

Wavenumber/cm

Figure 4. Room temperature bulk Raman (A) and surface-enhanced Raman (B and C) data from free amino acid (Trp). pH and molar concentration are reported. NaCl and KNO3 mean that ionic strength is increased either by 150 mM sodium chloride or 20 mM potatium nitrate, respectively. Characteristic Trp Raman markers as designated by W1-to-W8 are indicated in red color. The blue frame contains the two Trp SERS markers (Ws1 and Ws2) as marked by asterisks. 32


Page 33 of 38

A

B

@bulk

C

@Au/pH 3.5

@Au/pH 6/NaCl

D

@Au/pH 7/NaCl

10-6 M Ws1

1123

1235

Ws2

Y3 Y4 1209

Y3 Y4 1175

1230

Ws2

1175

Ws1

1208

1175

Y3 Y4

10-7 M 1122

Ws2

1209

Lanreotide

M

1128

Y3

Y4 1178

1235 1208

M

1113

10-7

1228

Ws1

10-2

Ws2 1119

Ws1 -5 Ws2 10 M 1232

Ws1

1118

10-5 M

1130

Ws2

1230

10-6 M

1113

10-2 M

1228

Ws1

1237

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


GWG 1300

1200

1100

1300

1200

1100

1300

1200

1100

1300

1200

1100

Wavenumber/cm -1

Figure 5. Room temperature bulk Raman (A) and surface-enhanced Raman data (B-D) of GWG (bottom row) and lanreotide (top row) recorded in the 1300-1150 cm-1 spectral region. pH and molar concentration are reported. NaCl means that ionic strength is increased by 150 mM sodium chloride. The positions of the two Trp SERS markers, Ws1 and Ws2, are also indicated in red color. 33



1384

1423

1600

10-5 M

1300

1700

1425

1620

1425 1400

1360 1344

1467 1344

1500

W5

W3 1540

1360

W4

1555

1601

1660 1700

1424

1534

W3

W5

W1 1481

1354 1472

1418

1700 1600 1500 1400 1300

1531

1521

1593 1555

10-6 M

W2

10-5 M W1

1681

1360 1340

1425 1460

1700 1600 1500 1400 1300

W5/W5

W2 W1

W4 W5

W2 W1 1620 1578

W3

1343

1360

10-6 M W1 W2 1619

W3

1559

W2

1469

1469 1420

10-7 M

W3

1552

GWG

W5 W4

W5

W4

W3

M

W4

1360 1343

1565

M

1678

Lanreotide

10-2

1524

W5

10-7

1670

1360 1344

1617

W1

Nal

W3 W4 1471 1441

M

1579 1552

W3 W2

@Au/pH 7/NaCl

Nal

Nal W4/W5

W2

D

@Au/pH 6/NaCl

1384 1356

1384

Nal

10-2

C

@Au/pH 3.5

1384

B

@bulk

1567 1531

A

1692

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 34 of 38

1600

1500

1400

1300

Wavenumber/cm-1

Figure 6. Room temperature bulk Raman (A) and surface-enhanced Raman data (B-D) of GWG (bottom row) and lanreotide (top row) recorded in the 1750-1300 cm-1 spectral region. pH and molar concentration are reported. NaCl means that ionic strength is increased by 150 mM sodium chloride.The positions of the five characteristic Trp markers, designated by W1-to-W5, are indicated. Y3 and Y4 refer to tyrosine markers, and Nal indicates a naphtylalanine Raman marker. 34


Page 35 of 38

A

B

@bulk

C

@Au/pH 3.5

D

@Au/pH 6/NaCl

@Au/pH 7/NaCl

771 760

1013

874

W8

10-5

M

W8 760

760

W7

W7 876

M

Y5 Y6

(Nal) W6 1012

10-5

NalW8 W7 853 830

874 1012 1002

755

W7

Y5 Y6

W6

W8

760

771

W7

Y6

872

M

Nal W8

876

874

W6

830

760

880

10-6

10-6 M (Nal)

W6

854

1013

996

Y5

W6

W8 W7

W7

996

880

852 828

Y5 Y6

W6

M

755

W8

Nal

854 830

760

10-7 M

Nal W6

W7

Lanreotide

10-2

10-7 M

1000

770

1017 1012

W8

Nal

1013

Nal

W6

10-2 M

770

Nal

1012

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


GWG 1000

900

800

700

1000

900

800

700

1000

900

800

700

1000

900

800

700

Wavenumber/cm-1

Figure 7. Room temperature bulk Raman (A) and surface-enhanced Raman data (B-D) of GWG (bottom row) and lanreotide (top row) recorded in the 1050-700 cm-1 spectral region. pH and molar concentration are reported. NaCl means that ionic strength is increased by 150 mM sodium chloride.The positions of the three characteristic Trp markers, designated by W6-to-W8, are indicated. Y5 and Y6 refer to tyrosine markers, and Nal indicates a naphtylalanine Raman marker. 35



ζ2

A

ε2

B

ε1

η2 ζ3

δ1

ε3

δ2

Page 36 of 38

ζ2 η2 ζ3

ε2

ε1 δ1

γ

ε3

δ2

γ

Cα αi-1 Cα αi

Cα αi-1 Cα αi

Cα αi-1

Cα αi-1

1130 cm-1

1230 cm-1 ν(Cε2-Nε1); ν(Nε1-Cδ1); δ(Cδ2-Cε3-H); ν(Cβ-Cγ)

δ(Cη2-Cζ2-H); ν(Cε3-Cζ3); δ(Cζ2-Cη2-H); ν(Cζ2-Cη2)

Figure 8. Graphical representation of the two calculated vibrational modes, supposed to be the precursors of the two Trp SERS markers (Ws1 and Ws2). Calculated modes are from the DFT calculation on the tripeptide Gly-Trp-Gly placed in a polarized solvent continuum (see text for details). The calculated wavenumber of each mode (scale factor=0.985), as well as its assignment in terms of internal coordinates are reported. For the sake of clarity the two extreme residues, i.e. Gly1 and Gly3 are only shown by their respective Cα atoms. 36


B

C

Decreasing pH

Decreasing pH

Na+

Increasing pH

Increasing pH

A

Decreasing pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Increasing pH

Page 37 of 38

D-Trp

Lys

Tyr

D-Nal

Nter

Figure 9. Suggested adsorption schemes for the three molecular compounds Trp (A), GWG (B) and lanreotide (C) on citrate-reduced gold nanoparticles. Molecular representations are made possible by means of the geometry optimized conformers displayed in Figure 1 (see text for details). Red and green arrows show the relative motion of the two interacting nanoparticles upon decreasing and increasing pH, respectively. 37



Page 38 of 38

TOC Graphic

38


Tryptophan Tight Binding to Gold Nanoparticles Induces Drastic

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