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A comparison between adsorption mechanism of tricyclic antidepressants on silver nanoparticles and binding modes on receptors. Surface-enhanced Raman spectroscopy studies
Journal of Colloid and Interface Science 431 (2014) 117–124
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
A comparison between adsorption mechanism of tricyclic antidepressants on silver nanoparticles and binding modes on receptors. Surface-enhanced Raman spectroscopy studies Aleksandra Jaworska, Kamilla Malek ⇑ Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland
a r t i c l e
i n f o
Article history: Received 10 March 2014 Accepted 29 May 2014 Available online 16 June 2014 Keywords: Tricyclic antidepressants Adsorption mechanism on Ag nanoparticles Surface-enhanced Raman spectroscopy (SERS) Drug action
a b s t r a c t A series of the tricyclic antidepressants known as a surface-active drugs, has been used as a model for an evaluation of their adsorption mechanism on the metal substrate and its relationship to pharmacological action of the chosen drugs. In these studies, six antidepressants were adsorbed on the metal substrate in a form of silver nanoparticles (ca. 30 nm in diameter) and afterwards their interactions have been examined in terms of surface-enhanced Raman spectroscopy (SERS). An analysis of SERS spectra has revealed that the dibenzopine moiety is a primary site of the adsorption with some differences in the orientation with respect to the metal among the studied molecules. The spectral changes due to the interactions with the silver particles also appear in the region typical for vibrations of the side chain. These observations are consistent with a model, in which the tricyclic ring is docked in the outer vestibule of biogenic amine transporters whereas the dimethyl-aminopropyl side chain is pointed to the substrate binding site. This work sheds a light on a potential of SERS technique in predicting a key functional group responsible for drug action. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Tricyclic antidepressants (TCAs) are the drugs commonly used to treat depression. Chemically, they are cationic, amphiphilic, small-size secondary or tertiary amines with a common core consisting of two flanking aromatic rings attached to a seven-membered ring. The latter can be a N- or O-heterocyclic ring (see Fig. 1). The most common TCAs are imipramine (Imi), desipramine (Des), clomipramine (Clo), amitriptyline (Ami), nortriptyline (Nor), and doxepine (Dox). They are categorized as selective serotonin, norepinephrine and dopamine reuptake inhibitors [1]. This type of the antidepressants still remains in clinical use, especially for treatment-resistant depression [2]. In toxic doses, however, they can lead to hypotension and cardiovascular disease and their use is associated with poisoning causing death. Thus, numerous analytical techniques such as fluorimetry, chemiluminometry, chromatography, enzyme and fluorescence polarization immunoassay and others [3 and therein] have been developed to identify and detect TCAs in biological matrix. The most often, their use is proceeded by an extraction the drugs from biological materials
⇑ Corresponding author. Fax: +48 12 634 0515. E-mail address: [email protected] (K. Malek). http://dx.doi.org/10.1016/j.jcis.2014.05.060 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
through liquid–liquid, solid phase, and microwave-assisted extraction techniques [4,5]. A potential application of surface-enhanced Raman spectroscopy (SERS) without a need of the extraction process has been also proposed [4]. Although tricyclic antidepressants have been used for years, their orientation within a primary target of their action has been yet unresolved. Sarker et al. have proposed a model, in which the tricyclic ring of the antidepressant is docked into the outer vestibule of serotonin transporter (SERT) whereas the drug’s dimethyl-aminopropyl side chain points to the substrate binding site. Consequently, such binding can create a structural change in the inner and outer vestibule, which precludes docking of the tricyclic ring [6]. In addition, studies on simultaneous binding of more than one antidepressant have indicated the presence of a second binding site in the inner vestibule, which can be the pseudo-symmetric fold of monoamine transporters [1]. Imipramine is a ligand that stabilizes SERT in the outward-facing conformation. Despite the fact that the flexibility of the methyl-aminopropyl side chain is restricted by a double bond in Ami, Nor and Dox, they adopt a docking pose similar to imipramine [6–8]. In studies of Sinning and co-workers on a series of structural analogues of imipramine, a salt bridge between the tertiary aliphatic amine of TCAs and Asp98 of the human serotonin transporter (hSERT) was identified while the 7-position of the imipramine ring was found vicinal to
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Fig. 1. Structures of the studied tricyclic antidepressants.
Phe335 [2]. A similar docking fashion of clomipramine into a transporter has been resolved in crystal structure of a complex between Clo and a biogenic amine transporter Leu-BAT [9]. In turn, crystal structure of the Drosophila melanogaster dopamine transporter (DAT) in complex with nortriptyline showed that the TCA along with cholesterol molecules stabilize the open conformation of the receptor through ionic interaction with one chloride and two sodium ions [8]. TCAs are also known as agents inducing lipidosis and intracellular accumulation of lipids. However, these interactions strongly depend on the type of the drug as well as a lipid structure and lipid phase transitions [10 and therein]. Optical-trapping confocal Raman spectroscopy has been used in the evaluation of the interaction of amitriptyline and nortriptyline with various phospholipid membranes [10]. Changes in an acyl chain conformation of the membranes and their intra- and intermolecular order due to the drugs action have been determined by monitoring peak intensities and positions of Raman signal originating from phospholipid vesicles. These studies have proposed that the TCAs interact via their cyclic rings with the acyl chains of the membranes whereas the tertiary aliphatic amine group is located near the lipid head groups. These results were found in agreement with pharmacological studies [10 and therein] and they have shown that Raman spectroscopy is an effective technique to elucidate drug–membrane interactions using micromolar concentrations of both lipids and drugs. The modern modification of Raman spectroscopy, surfaceenhanced Raman spectroscopy (SERS), utilizes generation of very strong electromagnetic field resulting from exciting of the localized surface plasmons in the metallic nanoparticles. SERS spectrum is observed if a molecule is in a close contact with a SERS-active support. Nowadays, SERS has been widely used in detection, identification and monitoring various biochemical processes since this technique has fast, label-free and non-invasive nature together with its high molecular specificity and sensitivity [11,12]. First of all, SERS provides valuable information on the adsorption mechanism of a (bio)molecule on a metallic surface pointing what functional groups or atoms participate in metal–adsorbate interactions.
In this study, we present SERS studies on six antidepressants: imipramine, desipramine, clomipramine amitriptyline, nortriptyline, and doxepine, whose structures are depicted in Fig. 1. We analyze the two groups of the tricyclic antidepressants: the first group contains the nitrogen atom inserted into the tricyclic ring (Imi, Des, Clo) and the second group represents the molecules with the double CC bond attached to the ring system (Ami, Nor, Dox). To record surfaceenhanced Raman signal of the molecules, colloidal silver particles were used and spectra were recorded with a laser excitation in the near-infrared region. The SERS spectra are analyzed in order to get insight into the adsorption behavior on the metal surface. Finally, we compare the proposed models of the surface adsorption of the chosen molecules with their docking profile into receptors. 2. Experimental 2.1. Chemicals All chemicals were purchased from Sigma, Germany and were of analytical grade. The TCAs were in a form of hydrochloride salt. Aqueous solutions of the antidepressants with the concentrations of 0.2 M (for normal Raman spectra) and 1 10 2 M (for SERS) were prepared by dilution of an appropriate amount of the analytes in the 4-fold distilled water. Silver colloid was prepared according to procedure described previously [13]. In this synthesis, Ag ions are reduced in the alkaline solution of hydroxylamine. UV–Vis spectrum of the silver colloid shows the presence of a resonant absorption band at ca. 412 nm (Fig. S1, Supporting Information). This position of the absorption maximum indicates that the size of silver nanoparticles varies in the range of 30–40 nm. Next, for SERS measurements, 10 lL of a sample solution was mixed with 500 lL of a colloid. The final concentration of the analytes in the mixture was 2 10 4 M. 2.2. Instrumentation The absorption spectra were recorded with a UV–Vis–NIR Nicolet spectrophotometer (model Evolution 60) in the range of
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190–1100 nm with a resolution of 1 nm. To place a sample, quartz cells of 1 cm width were used. All Raman spectra were recorded on a MultiRAM FT-Raman Spectrometer (Bruker) equipped with a Nd:YAG laser, emitting at 1064 nm, and a germanium detector cooled with liquid nitrogen. The output power of the laser was 150–300 mW while the spectral resolution was 4 cm 1 for all measurements. 128, 4000, and 1024 Scans were collected for the solids, solutions and SERS, respectively. A few milligrams of solids and 1 mL of solutions were placed on metal discs and in quartz cuvettes, respectively. Integrated intensities of the chosen bands were calculated from the Raman and SERS spectra employing integration method. To accomplish this, a linear baseline was drawn through the peak edges, and the spectrum below this line was integrated over the wavenumber range of the band. All spectra processing was performed by using a Bruker OPUS software (Version 6.5). 2.3. An assignment of Raman bands Density functional theory (DFT) calculations were carried out for geometry optimization and simulation of Raman spectra. These calculations were performed with the Gaussian 09 program package [14] by using B3LYP method [15,16] and 6-31+G(d) basis set. No imaginary frequencies were obtained. Next, theoretical Raman intensities (IR) were obtained from Gaussian Raman scattering activities (S) according to the expression in [17]. Then, simulated spectra were compared to experimental normal Raman spectra of the solids (Figs. S2–S7, Supporting Information). To provide the unequivocal assignment of the calculated Raman spectra, the potential energy distribution (PED) analysis was performed by using the Gar2ped software [18]. The program first defines a set of non-redundant internal coordinates according to the Pullay’s and Foragasi’s definitions [19,20], and next the percentage contribution of the internal coordinates to the total energy of each normal mode is computed. Definitions of the internal coordinates used in this work are collected in Table S1 (Supporting Information).
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In our experiment, SERS spectra were recorded in the presence of potassium chloride as an aggregating agent as well as without it. However, despite the fact that halide ions generally increase SERS intensity due to the formation of Ag+–Cl -ligand surface complexes or due to an increase of the local electromagnetic field by aggregation of metal colloid particles, we observed a decrease of SERS signal of the antidepressants by ca. 20–30% after addition of KCl (data not shown). This indicates that SERS phenomenon for TCAs is negatively affected by KCl because of adsorption competition between chlorides ions and the adsorbate due to the formation of Ag–Cl clusters on the metal surface [4,21]. Figs. 2 and 3 display the normal Raman (NR) and SERS spectra of the antidepressants containing the nitrogen atom in the 7-membered ring and the C@C bond attached to this ring, respectively. Since Raman bands of the antidepressants are not observed in spectra of the solutions at the concentration of 2 10 4 M used in SERS, the SERS spectra shown in Figs. 2 and 3 are exclusively due to surface enhancement of the Raman scattering of adsorbed molecules by silver nanoparticles. Firstly, a close analysis of NR spectra of solutions (Figs. 2 and 3) and solids (Figs. S2–S7) reveals no substantial differences besides the well-known characteristics of the solution spectra concerning the broadening and slight changes in the peak position of the bands. Thus, Tables 1–6 summarize the assignment of the Raman and SERS bands of the solutions on the basis of DFT calculations described above while Tables 7 and 8 summarize the selected relative intensities of bands originating from the phenyl and 7-membered rings, the aliphatic chain and the terminal methyl groups. Instead of a complete comparison of all modes of all the molecules, we will focus on a few regions of the Raman and SERS spectra, which can be of a special interest in terms of the adsorption mechanism on the silver
3. Results and discussion UV–Vis absorption spectra of the silver colloid and its mixture with the adsorbate point out what type of SERS mechanism plays a significant role in the adsorption process on a metal colloid. The presence of a single band due to particle plasmon resonance indicates physisorption of a molecule on the sol whereas the appearance of the second peak in the red/near-infrared spectral region suggests a contribution of charge transfer mechanism due to the molecule–metal interaction. UV–Vis spectrum of the silver colloid used in this work exhibits the presence of the plasmon resonance band at ca. 412 nm typical for this sol (Fig. S1B, Supporting Information). After addition of the solution of the drug, absorbance of this band significantly decreases along with blue-shifting of the maximum (Fig. S1C). Broadening of the band indicates the aggregation of the silver particles due to the interaction of the analyte with the silver. Fig. S1C shows an exemplary spectrum for imipramine, however, the spectra of the other TCAs exhibit similar features. In addition, the second plasmon resonance band is observed at ca. 900 nm. This suggests that charge-transfer mechanism may also contribute to surface-enhancement of Raman signal recorded with the use of the 1064 nm laser excitation. For the analytical purpose, we also investigated SERS features of Imi and Des by using a laser excitation at 785 nm [4]. In general, band positions and relative intensities are very similar for both NIR wavelengths of exciting light, indicating a similar mechanism of enhancement of Raman signal.
Fig. 2. Normal (c = 0.2 M) and SERS (c = 2.0 10 4 M) spectra of imipramine (A and B, respectively), desipramine (C and D, respectively) and clomipramine (E and F, respectively).
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A. Jaworska, K. Malek / Journal of Colloid and Interface Science 431 (2014) 117–124 Table 2 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of desipramine in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
411a 495 565 604 685 779 973 1044 1063 1127 1165 1208 1233 1438 1475 1598 2957a 3051a
423 498 565 621 683 774 970 1042 1059 1127 1161 1206 1231 1433 1470 1595 2963 3055
sR6 (29), bR6 (16), bflR62/R7 (9) sR6 (18), sR7 (15), bR6 (8), xN1C/aliph (8) bR7 (33), bR6 (22), xNC/aliph (9) sR6 (42), bR7 (13) bR6 (34), mCC/R7 (19), sR6 (7) sR6 (15), xN2H (8), bR6 (7), mCC/R7 (7) mCC/R7 (32), qCH2/R7 (25) mCC/aliph (32), mCC/R62 (21), mCC/R61 (53), qCH/R61 (12) mNC/aliph (13), bR7 (9), sCH2/R7 (7) qCH/R61 (13), qCH/R62 (9) mCC/R7 (33), bR6 (13), mCC/R62 (7) mNC/R7 (18), qCH/R61 (12), sCH2/R7 (11), mNC/aliph (7) dCH2/aliph (83) dCH2/R7 (80) mCC/R61 (31), mCC/R62 (23) mCH/met (89) mCH/R62 (86)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, btf – butterfly, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs – very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1. a Observed in the Raman spectrum of the solid sample.
Fig. 3. Normal (c = 0.2 M) and SERS (c = 2.0 10 4 M) spectra of amitriptyline (A and B, respectively), nortriptyline (C and D, respectively) and doxepine (E and F, respectively).
Table 1 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of imipramine in the range of 500–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
389 450 498 567 685 973 1044 1063 1127 1207 1232 1436 1473 1597 2971 3062
386 432 500 567 683 972 1042 1061 1127 1206 1229 1431 1468 1597 2963 3057
xNC/R7 (26), sR7 (15), qNC/aliph (7) bNC/aliph (35), sR6 (16), xNC/R7 (7) dCH2/aliph (36), qNC (11) bR7 (23), bR6 (22), xN1C (9) bR6 (32), mCC//R7 (19), sR6 (7) mCC/R7 (32), qCH2/R7 (25) mCC/aliph (38), dCH2/aliph (21), bNC/met (12) mCC/R61 (41), mNC/aliph (8) mNC/aliph (14), bR7 (10), sCH2/R7 (8), mCC/R62 (7) mCC/R7 (33), bR6 (13) mNC/R7 (13), sCH2/aliph (10), mNC/aliph (7), qCH2/aliph (7) bmet (46), dCH2/aliph (42) dCH2/aliph (38), bmet (31) mCC/R62 (35), mCC/R61 (31) mCH/met (89) mCH/R62 (96)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, R6 – 6membered ring, R7 – 7-membered ring, aliph – aliphatic chain, Met – methyl group, vs –very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
nanoparticles. At first glance, the SERS spectra exhibit the presence of all bands observed in the corresponding NR spectra. No shift and significant broadening of SERS bands are observed, thus electromagnetic (long-distance) mechanism of surface enhancement of Raman signal rather plays a leading role in SERS of TCAs. However, the most pronounced feature of all the SERS spectra is a strong enhancement of a band at ca. 680–690 cm 1 attributed to the inplane bending mode of both the flanking phenyl rings (bR6) coupled
Table 3 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of clomipramine in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
370 399 462 507 557 631 692 769 974 1044 1065 1105 1127 1165 1208 1231 1437 1592 2974 3064a
372 399 463 506 556 629 692 770 972 1044 1061 1101 1128 1161 1206 1229 1431 1591 2960 3054
sR6 (36), bR7 (16), bR6 (10) bR7 (30), bR6 (21), mCCl (10) bCN/met (22), sR6 (13) dCH2/aliph (34), sR6 (15), qNC (11), bR7 (7) bR6 (17), bR7 (14), sR7 (10), xNC/aliph (9) sR6 (33), xCCl (21), bR6 (8) bR6 (39), mCC/R7 (18) xCH/R62 (33), bR6 (10) mCC/R7 (32), qCH2/R7 (26) mCC/aliph (36), dCH2/aliph (21), bNC/met (15) mCC/R61 (36), qCH/R61 (17) mCC/R62 (28), b/R6 (7) mCC/R61 (16), qCH/R61 (15), mCC/R62 (11) qCH/R61 (85) mCC/R7 (33), b/R6 (13) mNC/R7 (20), mNC/aliph (11), sCH2/R7 (9) dCH2/aliph (89)
mCC/R62 (41), mCC/R61 (12), mCC/R7 (7) mCH/met (86), mCH/aliph (8) mCH/R62 (97)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs – very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1. a Observed in the Raman spectrum of the solid sample.
with the stretches of the CAC bonds of the 7-membered ring. This mode is a simultaneous in-phase breathing vibration of the dibenzazepine ring. We compare the enhancement of this mode with SERS intensity of the phenyl ring CC stretching vibration (mCCR6) observed at 1600 cm 1 and often denoted as the 8a mode (according to the Wilson’s notation, [22], see PED in Tables 1–6). Since the former is a symmetric vibration, its large enhancement due to the adsorption on the metal surface confirms its origin from electromagnetic rather than from charge-transfer mechanism of the SERS phenomenon [23]. First of all, the enhancement of the 680 cm 1 band gives an evidence for a strong interaction of the conjugated
A. Jaworska, K. Malek / Journal of Colloid and Interface Science 431 (2014) 117–124 Table 4 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of amitriptyline in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
334 534 689 966 1040 1055 1163 1208 1368 1431 1599 1638 2972 3047
336 534 689 968 1038 1053 1163 1208 1366 1427 1597 1638 2963 3042
sR6 (28), bR7 (25) bR6 (28), bR7 (12), sR6 (7), mCC/R7 (7) bR6 (48), mCC/R7 (7) mCC/R7 (29), qCH2/R7 (25), bR61(7) mCC/aliph (56), qCH/R62 (13) mCC/R61 (56), qCH/R61 (12) mCC/R7 (16), mCC/R62 (12), bR6 (11), qCH/R61 (9) mCC/R7 (25), qCH/R62 (12), mCC/R62 (10), bR6 (7) qCH2/aliph (52), mCC/R7 (9) bNC/met (95) mCC/R61 (53), qCH/R61 (13), bR6 (8) mC@C (64), qCH/aliph (9), mCC/aliph (7) mCH/met (94) mCH/R62 (99)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs – very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
Table 5 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of nortriptyline in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
334 534 690 970 1040 1055 1165 1208 1369 1425 1599 1641 2963 3052
336 535 689 968 1038 1057 1163 1206 1370 1427 1597 1641 2963 3044
sR6 (32), bR7 (26) bR6 (27), sR6 (8), mCC/R7 (7), bR7 (7) bR6 (46), mCC/R7 (8) mNC/aliph (29), mNC/met (21), bNC/met (11) mCC/aliph (68), qCH/R62 (15) mCC/R61 (60), qCH/R61 (12) bNC/met (40), mNC/aliph (27) mCC/R7 (30), qCH/R62 (11), mCC/R62 (10), bR6 (7) qCH2/aliph (45), mCC/R7 (10), xCH2/R7 (8) bNC/met (86)
mCC/R62 (54), qCH/R62 (13) mC@C (64), qCH/aliph (9), mCC/aliph (7) mCH/R7 (97) mCH/R62 (95)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs –very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
Table 6 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of doxepine in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
343 455 696 838 1041 1050 1165 1221 1369 1461 1604 1638 2960 3060
343 457 696 837 1040 1051 1157 1221 1366 1459 1601 1636 2961 3056
sR6 (32), bR7 (22) qNC/met (23), bR61(10) bR6 (44), mCC/R7 (17), mCC/R7 (8) mNC/met (48), mNC/aliph (20) mCC/aliph (42), mOC/R7 (24), mOC (31), mCC/R62 (24), qCH2/R7 (8) qCH/R62 (45), mCC/R62 (10) mOC (28), qCH/R62 (10), mCC/R7 (10) qCH2/aliph (46), mCC/R7 (14) bCH3 (82)
mCC/R61 (31), mCC/R62 (19) mC@C (63), qCH/aliph (9) mCH/met (96) mCH/R61 (95)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs –very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
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system of the rings and the silver surface instead of the most common adsorption on the silver nanoparticles via the p-electron system of the aromatic rings [24–26]. In addition, weakening of the 8a mode and the presence of very weak bands originating from the stretching vibration of the aromatic CH bonds (about 3055 cm 1, cf. Figs. 2 and 3) confirm that the phenyl rings are not oriented perpendicularly to the metal surface [27]. Thus, we conclude that the conjugated rings are tilted with the respect to the metal adopting an edge-on geometry. The detailed examination of the SERS spectra of the drugs containing the nitrogen atom in the 7-membered ring reveals that their SERS features differ even due to small structural modifications such as substitution of the methyl group by the hydrogen atom in imipramine and desipramine (Imi, Des, Clo; cf. Figs. 1 and 2). The mentioned-above ratio of bands at 685 and 1600 cm 1 (I683/I1600) in these SERS spectra increases noticeably relative to the corresponding normal Raman bands by a factor of 2–8, confirming the contribution of the dibenzazepine system in the interaction with the silver (Table 7). This ratio in the SERS spectra is similar for imipramine and clomipramine whereas it is a slightly lower for desipramine (see Table 7). Since spatial arrangement of the ring systems in all the antidepressants is similar whereas the I683/I1600 ratio is similar for Imi and Clo containing the chlorine atom in the phenyl ring, we assumed that the loss of the methyl group in the aliphatic chain of desipramine may result in the competition of the other fragment of this antidepressant in the adsorption process and/or a different orientation of its rings with respect to the silver surface. In the case of the latter, we notice variation in the ratio between bands at 683 and 1060 cm 1 (cf. Table 7). According to PED calculations for this group of antidepressants, the 1060 cm 1 band is assigned to the in-phase stretches of the CC bonds of the R61 ring that are non-conjugated to the 7-membered ring (see Fig. 1 for numbering). The I683/I1060 ratio is much lower for desipramine than for imipramine and clomipramine indicating a larger enhancement of the 1060 cm 1 band for Des than for Imi and Clo due to the adsorption on the silver sol. This can suggest that one of the phenyl rings of Des is directed towards the silver nanoparticles whereas Imi and Clo interact through the entire dibenzazepine moiety. Additionally, among the molecules discussed here, the out-of-plane bending modes of the rings observed below 650 cm 1 are enhanced in the SERS spectra of Imi and Des, see Fig. 2 and Tables 1 and 2. They are also present in the SERS spectrum of clomipramine but their intensities are similar to those in the normal Raman spectrum of the solution. This may suggest that the interaction of the Imi and Des aromatic rings with the metal via the p-electron system also appears. Hence, the presence of these modes and the in-plane bending vibrations of the rings indicates a slightly tilted orientation of the dibenzazepine rings of Imi and Des in comparison to Clo. We also examine the change in the integral intensity of the marker bands of the aliphatic chain, i.e. mCC and dCH2 observed at 1042 and 1431 cm 1, respectively, with respect to the 683 cm 1 band (see Table 7). Among the three antidepressants, the SERS spectrum of desipramine exhibits the largest surface enhancement of the mCCaliph mode along with a very low increase in the intensity of the scissoring vibration of the methylene groups. The opposite trend is found for clomipramine. Hence we concluded that the aliphatic chain of Des is oriented almost perpendicularly with respect to the metal whereas it is strongly tilted in clomipramine. In the case of imipramine, the aliphatic chain adopts a intermediate geometry between those proposed for Des and Clo. These orientations of the side chain are also confirmed by the SERS ratio of the 1042 and 1431 cm 1 bands, see Table 7. The second group of the tricyclic antidepressants studied here, i.e. amitriptyline, nortriptyline and doxepine, characterize the presence of the double CC bond attached to the dibenzazepine system,
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Table 7 Relative intensities of selected bands in NR and SERS spectra of imipramine (Imi), desipramine (Des) and clomipramine (Clo).
a b c d
I683/I1600a
I683/I1060b
I689/I1042c
I689/I1431d
I1042/I1431
Imi
NR SERS
0.5 2.2
2.9 11.5
1.6 5.8
7.0 3.4
4.4 0.6
Des
NR SERS
0.7 1.4
2.6 7.8
1.2 4.0
5.7 5.0
4.7 1.2
Clo
NR SERS
0.3 1.9
4.2 11.9
3.1 8.8
2.4 2.0
0.8 0.2
683 cm 1: bR6/mCCR7, 1600 cm 1060 cm 1: mCCR61. 1042 cm 1: mCCaliph. 1431 cm 1: dCH2.
1
: mCCR6.
Table 8 Relative intensities of bands in NR and SERS spectra of amitriptyline (Ami), nortriptyline (Nor) and doxepine (Dox).
a b c d
I690/I1600a
I690/I1640b
I690/I1042c
I690/I1369d
I1042/I1369
Ami
NR SERS
0.6 4.7
0.4 1.9
2.0 6.1
2.9 8.5
1.5 1.4
Nor
NR SERS
0.5 3.1
0.3 1.2
2.0 4.5
2.3 3.8
1.2 0.8
Dox
NR SERS
0.6 3.5
0.4 2.0
3.7 5.5
1.7 4.0
0.5 0.7
690 cm 1: bR6; 1600 cm 1640 cm 1: mC@C. 1042 cm 1: mCCaliph. 1369 cm 1: qCH2/aliph.
1
: mCCR6.
Fig. 4. The proposed model of the adsorption mechanism of the antidepressants (colours of atoms correspond to: gray – carbon, white – hydrogen, blue – nitrogen, green – chlorine, red – oxygen). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cf. Fig. 1. The detailed assignment of Raman and SERS bands is collected in Tables 4–6. Similarly to the group discussed above, the SERS spectra of those antidepressants are similar to the NR spectra in means of bands positions and they also exhibit the enormous intensification of a band at 689 and 696 cm 1 for Ami/Nor and Dox, respectively (Fig. 3). First of all, the I690/I1600 ratio increases 6–8 times in the SERS spectra in comparison to the Raman spectra of the solutions like for the previous group of the molecules, confirming that the dibenzazepine moiety is a primary site of the
interaction with the surface. Interestingly, this ratio is similar for SERS of nortriptyline and doxepine, despite the presence of the oxygen atom in the 7-membered ring of Dox, but the largest value of the ratio is found for amitryptiline. Thus, nortryptiline and doxepine adopt a less tilted orientation of the ring system in comparison to amitryptiline since the 8a mode of the phenyl ring is more enhanced than the bending motion of the three rings together. Moreover, according to our PED calculations, the out-of-plane bending vibration of the phenyl rings conjugated with the in-plane
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bending mode of the R7 ring is manifested by the presence of a weak band at ca. 340 cm 1 (Tables 4–6), which becomes a medium-intensity band in the SERS spectra of all the molecules. Therefore, one can assume that, for this group of the antidepressants, the molecules are oriented in such a way that the benzene rings are preponderantly tilted with respect to the surface. Along with decreasing the intensity of the 8a mode of the phenyl rings due to adsorption on the silver nanoparticles, we also observed lowering intensity of the stretching vibration of the C@C bond (at 1640 cm 1) comparing to the corresponding normal Raman spectra. The intensity ratio of these bands (I690/I1640) in the NR spectra is similar for all molecules indicating significantly larger intensity of the C@C stretching mode than the breathing vibration of the dibenzazepine group. After adsorption on the metal, the latter has an intensity twice as large as the mC@C band in the spectra of Ami and Dox and slightly larger for Nor (cf. Table 8). The molecular structures of the antidepressants studied here rather exclude a simultaneous perpendicular orientation of the dibenzazepine moiety and the C@C bond with respect to the surface (cf. Fig. 4). Thus the ratio between the 690 and 1640 cm 1 bands can indicate to what extend both functional groups are directed perpendicularly to the metal. This ratio is similar for Ami and Dox (ca. 2) whereas it decreases to 1.2 for nortriptyline indicating that the double CC bond is reoriented from a tilted to an upright geometry, respectively. An analysis of the SERS bands attributed to vibrations of the aliphatic chain, mCC (1042 cm 1) and qCH2 (1369 cm 1), exhibits a similar enhancement of the mCC mode with respect to the 690 cm 1 band for all the antidepressants whereas a 2-fold decrease of the I690/I1369 ratio is found for Nor and Dox in comparison to the ratio values calculated for amitriptyline. In turn, the relative intensities of the 1042 and 1369 cm 1 bands clearly indicate an orientation of the side on the silver nanoparticles (see Table 8). Since the I1042/I1369 ratio is similar in the NR and SERS spectra of Ami and Dox, the aliphatic chain is likely oriented in a way similar to the DFT-optimized structures (cf. Fig. 4 and Table 8). In turn, the SERS intensity of the dCH2 mode is higher than the intensity of the mCC vibration for Nor, suggesting that the aliphatic chain of Nor is more tilted to the surface than for Ami and Dox. The reason for that could be the absence of one methyl group in Nor and in consequence a stronger interaction of the lone electron pair of the terminal N atom than in the other molecules. However, the opposite effect is observed for desipramine as discussed above and this clearly results from the different orientations of the dibenzazepine moiety on the silver surface. Summarizing the proposed adsorption models for all antidepressants studied here are illustrated in Fig. 4.
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identified the most favorable location. However, all of them suggest that the tricyclic ring adopts a tilted orientation with respect to the hydrophilic pocket of the serotonin transporter similarly to the TCAs interaction with the metal surface. The possible rotational reorientation of 3-substituted antidepressants like clomipramine from the symmetric docking of imipramine in hSERT could be in the agreement with our SERS spectra that revealed a less tilted orientation of the tricyclic moiety of Clo. A similar orientation of the ring system of nortriptyline was identified in its SERS spectrum that is congruent with the fact that Nor is wedged between transmembrane helices of the dopamine transporter [8]. No detailed data on locations of amitriptyline and doxepin within a transporter have been reported so far in the literature, thus a direct comparison between the SERS model of the adsorption on the metal surface and pharmacological action of the two antidepressants is limited. On the other hand, the proposed models of the surface adsorption for Ami and Dox (Fig. 4) suggest a greater preferential interaction between the p-electron system of the azepine ring and silver nanoparticles in comparison to that observed for the ring system of nortriptyline. Moreover, SERS features of Ami and Dox can implicate that the presence of the oxygen atom in the azepine ring enforces slightly different strength and/or binding fashion with the metallic surface through this moiety. It is also worth mentioning that the binding affinity studies of TCAs have suggested a contribution of chloride ions into the stabilization of their docking pose for a given receptor conformation and they have assumed the presence of one chloride ion in the close vicinity of the antidepressant molecule [2,9]. In turn, in our SERS investigations, we observed weakening of the adsorbate–metal interactions when an excess of chloride ions was added to the mixture of antidepressants and silver sol, taking into account that the TCA:Cl molar ratio of 1:1 is already introduced to the solution. Our results demonstrate an analogy between the molecular structure and orientation of the investigated drugs when interacting with the colloidal silver nanoparticles and their orientation within the central substrate site of the transporters. This suggests, despite the simplicity of the model, that SERS can serve as a probing and complimentary method for experimental pharmacological studies and molecular dynamics simulations. Although, it should be also pointed out that surface-enhanced Raman spectroscopy does not allow identifying the specific amino acid residues as binding sites in the receptors and the type of interaction like p–p stacking, hydrogen bonding or water-mediated salt bridge, but this may rather mimic a docking pose of a ligand within a receptor. Appendix A. Supplementary material
4. Conclusions In this work, we present the orientation of the six tricyclic antidepressants on the silver nanoparticles determined by surface-enhanced Raman spectroscopy. We identified the primary adsorption site on the silver nanoparticles via the p-electron system of the ring moiety and the methyl-aminopropyl side chain. The orientation of the dibenzazepine ring slightly differs between the molecules pointing an effect of the complex structure of the molecules on the subtle changes in their surface behavior. These changes in the orientation of the ring systems are consistent with the interactions of TCAs within the central substrate site of the serotonin and leucine transporters in that the ring system is lying almost perpendicularly in the membrane plane. The alkylamine side chain is oriented similarly to the ring, however, according to molecular dynamics simulations its spatial arrangement varies for the particular antidepressant [2]. Several works cited in Section 1 [2,6,8,9,28] suggest that the position of the particular TCA is flexible in the binding site and in some cases it is difficult to
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
A comparison between adsorption mechanism of tricyclic antidepressants on silver nanoparticles and binding modes on receptors. Surface-enhanced Raman spectroscopy studies Aleksandra Jaworska, Kamilla Malek ⇑ Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland
a r t i c l e
i n f o
Article history: Received 10 March 2014 Accepted 29 May 2014 Available online 16 June 2014 Keywords: Tricyclic antidepressants Adsorption mechanism on Ag nanoparticles Surface-enhanced Raman spectroscopy (SERS) Drug action
a b s t r a c t A series of the tricyclic antidepressants known as a surface-active drugs, has been used as a model for an evaluation of their adsorption mechanism on the metal substrate and its relationship to pharmacological action of the chosen drugs. In these studies, six antidepressants were adsorbed on the metal substrate in a form of silver nanoparticles (ca. 30 nm in diameter) and afterwards their interactions have been examined in terms of surface-enhanced Raman spectroscopy (SERS). An analysis of SERS spectra has revealed that the dibenzopine moiety is a primary site of the adsorption with some differences in the orientation with respect to the metal among the studied molecules. The spectral changes due to the interactions with the silver particles also appear in the region typical for vibrations of the side chain. These observations are consistent with a model, in which the tricyclic ring is docked in the outer vestibule of biogenic amine transporters whereas the dimethyl-aminopropyl side chain is pointed to the substrate binding site. This work sheds a light on a potential of SERS technique in predicting a key functional group responsible for drug action. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Tricyclic antidepressants (TCAs) are the drugs commonly used to treat depression. Chemically, they are cationic, amphiphilic, small-size secondary or tertiary amines with a common core consisting of two flanking aromatic rings attached to a seven-membered ring. The latter can be a N- or O-heterocyclic ring (see Fig. 1). The most common TCAs are imipramine (Imi), desipramine (Des), clomipramine (Clo), amitriptyline (Ami), nortriptyline (Nor), and doxepine (Dox). They are categorized as selective serotonin, norepinephrine and dopamine reuptake inhibitors [1]. This type of the antidepressants still remains in clinical use, especially for treatment-resistant depression [2]. In toxic doses, however, they can lead to hypotension and cardiovascular disease and their use is associated with poisoning causing death. Thus, numerous analytical techniques such as fluorimetry, chemiluminometry, chromatography, enzyme and fluorescence polarization immunoassay and others [3 and therein] have been developed to identify and detect TCAs in biological matrix. The most often, their use is proceeded by an extraction the drugs from biological materials
⇑ Corresponding author. Fax: +48 12 634 0515. E-mail address: [email protected] (K. Malek). http://dx.doi.org/10.1016/j.jcis.2014.05.060 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
through liquid–liquid, solid phase, and microwave-assisted extraction techniques [4,5]. A potential application of surface-enhanced Raman spectroscopy (SERS) without a need of the extraction process has been also proposed [4]. Although tricyclic antidepressants have been used for years, their orientation within a primary target of their action has been yet unresolved. Sarker et al. have proposed a model, in which the tricyclic ring of the antidepressant is docked into the outer vestibule of serotonin transporter (SERT) whereas the drug’s dimethyl-aminopropyl side chain points to the substrate binding site. Consequently, such binding can create a structural change in the inner and outer vestibule, which precludes docking of the tricyclic ring [6]. In addition, studies on simultaneous binding of more than one antidepressant have indicated the presence of a second binding site in the inner vestibule, which can be the pseudo-symmetric fold of monoamine transporters [1]. Imipramine is a ligand that stabilizes SERT in the outward-facing conformation. Despite the fact that the flexibility of the methyl-aminopropyl side chain is restricted by a double bond in Ami, Nor and Dox, they adopt a docking pose similar to imipramine [6–8]. In studies of Sinning and co-workers on a series of structural analogues of imipramine, a salt bridge between the tertiary aliphatic amine of TCAs and Asp98 of the human serotonin transporter (hSERT) was identified while the 7-position of the imipramine ring was found vicinal to
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Fig. 1. Structures of the studied tricyclic antidepressants.
Phe335 [2]. A similar docking fashion of clomipramine into a transporter has been resolved in crystal structure of a complex between Clo and a biogenic amine transporter Leu-BAT [9]. In turn, crystal structure of the Drosophila melanogaster dopamine transporter (DAT) in complex with nortriptyline showed that the TCA along with cholesterol molecules stabilize the open conformation of the receptor through ionic interaction with one chloride and two sodium ions [8]. TCAs are also known as agents inducing lipidosis and intracellular accumulation of lipids. However, these interactions strongly depend on the type of the drug as well as a lipid structure and lipid phase transitions [10 and therein]. Optical-trapping confocal Raman spectroscopy has been used in the evaluation of the interaction of amitriptyline and nortriptyline with various phospholipid membranes [10]. Changes in an acyl chain conformation of the membranes and their intra- and intermolecular order due to the drugs action have been determined by monitoring peak intensities and positions of Raman signal originating from phospholipid vesicles. These studies have proposed that the TCAs interact via their cyclic rings with the acyl chains of the membranes whereas the tertiary aliphatic amine group is located near the lipid head groups. These results were found in agreement with pharmacological studies [10 and therein] and they have shown that Raman spectroscopy is an effective technique to elucidate drug–membrane interactions using micromolar concentrations of both lipids and drugs. The modern modification of Raman spectroscopy, surfaceenhanced Raman spectroscopy (SERS), utilizes generation of very strong electromagnetic field resulting from exciting of the localized surface plasmons in the metallic nanoparticles. SERS spectrum is observed if a molecule is in a close contact with a SERS-active support. Nowadays, SERS has been widely used in detection, identification and monitoring various biochemical processes since this technique has fast, label-free and non-invasive nature together with its high molecular specificity and sensitivity [11,12]. First of all, SERS provides valuable information on the adsorption mechanism of a (bio)molecule on a metallic surface pointing what functional groups or atoms participate in metal–adsorbate interactions.
In this study, we present SERS studies on six antidepressants: imipramine, desipramine, clomipramine amitriptyline, nortriptyline, and doxepine, whose structures are depicted in Fig. 1. We analyze the two groups of the tricyclic antidepressants: the first group contains the nitrogen atom inserted into the tricyclic ring (Imi, Des, Clo) and the second group represents the molecules with the double CC bond attached to the ring system (Ami, Nor, Dox). To record surfaceenhanced Raman signal of the molecules, colloidal silver particles were used and spectra were recorded with a laser excitation in the near-infrared region. The SERS spectra are analyzed in order to get insight into the adsorption behavior on the metal surface. Finally, we compare the proposed models of the surface adsorption of the chosen molecules with their docking profile into receptors. 2. Experimental 2.1. Chemicals All chemicals were purchased from Sigma, Germany and were of analytical grade. The TCAs were in a form of hydrochloride salt. Aqueous solutions of the antidepressants with the concentrations of 0.2 M (for normal Raman spectra) and 1 10 2 M (for SERS) were prepared by dilution of an appropriate amount of the analytes in the 4-fold distilled water. Silver colloid was prepared according to procedure described previously [13]. In this synthesis, Ag ions are reduced in the alkaline solution of hydroxylamine. UV–Vis spectrum of the silver colloid shows the presence of a resonant absorption band at ca. 412 nm (Fig. S1, Supporting Information). This position of the absorption maximum indicates that the size of silver nanoparticles varies in the range of 30–40 nm. Next, for SERS measurements, 10 lL of a sample solution was mixed with 500 lL of a colloid. The final concentration of the analytes in the mixture was 2 10 4 M. 2.2. Instrumentation The absorption spectra were recorded with a UV–Vis–NIR Nicolet spectrophotometer (model Evolution 60) in the range of
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190–1100 nm with a resolution of 1 nm. To place a sample, quartz cells of 1 cm width were used. All Raman spectra were recorded on a MultiRAM FT-Raman Spectrometer (Bruker) equipped with a Nd:YAG laser, emitting at 1064 nm, and a germanium detector cooled with liquid nitrogen. The output power of the laser was 150–300 mW while the spectral resolution was 4 cm 1 for all measurements. 128, 4000, and 1024 Scans were collected for the solids, solutions and SERS, respectively. A few milligrams of solids and 1 mL of solutions were placed on metal discs and in quartz cuvettes, respectively. Integrated intensities of the chosen bands were calculated from the Raman and SERS spectra employing integration method. To accomplish this, a linear baseline was drawn through the peak edges, and the spectrum below this line was integrated over the wavenumber range of the band. All spectra processing was performed by using a Bruker OPUS software (Version 6.5). 2.3. An assignment of Raman bands Density functional theory (DFT) calculations were carried out for geometry optimization and simulation of Raman spectra. These calculations were performed with the Gaussian 09 program package [14] by using B3LYP method [15,16] and 6-31+G(d) basis set. No imaginary frequencies were obtained. Next, theoretical Raman intensities (IR) were obtained from Gaussian Raman scattering activities (S) according to the expression in [17]. Then, simulated spectra were compared to experimental normal Raman spectra of the solids (Figs. S2–S7, Supporting Information). To provide the unequivocal assignment of the calculated Raman spectra, the potential energy distribution (PED) analysis was performed by using the Gar2ped software [18]. The program first defines a set of non-redundant internal coordinates according to the Pullay’s and Foragasi’s definitions [19,20], and next the percentage contribution of the internal coordinates to the total energy of each normal mode is computed. Definitions of the internal coordinates used in this work are collected in Table S1 (Supporting Information).
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In our experiment, SERS spectra were recorded in the presence of potassium chloride as an aggregating agent as well as without it. However, despite the fact that halide ions generally increase SERS intensity due to the formation of Ag+–Cl -ligand surface complexes or due to an increase of the local electromagnetic field by aggregation of metal colloid particles, we observed a decrease of SERS signal of the antidepressants by ca. 20–30% after addition of KCl (data not shown). This indicates that SERS phenomenon for TCAs is negatively affected by KCl because of adsorption competition between chlorides ions and the adsorbate due to the formation of Ag–Cl clusters on the metal surface [4,21]. Figs. 2 and 3 display the normal Raman (NR) and SERS spectra of the antidepressants containing the nitrogen atom in the 7-membered ring and the C@C bond attached to this ring, respectively. Since Raman bands of the antidepressants are not observed in spectra of the solutions at the concentration of 2 10 4 M used in SERS, the SERS spectra shown in Figs. 2 and 3 are exclusively due to surface enhancement of the Raman scattering of adsorbed molecules by silver nanoparticles. Firstly, a close analysis of NR spectra of solutions (Figs. 2 and 3) and solids (Figs. S2–S7) reveals no substantial differences besides the well-known characteristics of the solution spectra concerning the broadening and slight changes in the peak position of the bands. Thus, Tables 1–6 summarize the assignment of the Raman and SERS bands of the solutions on the basis of DFT calculations described above while Tables 7 and 8 summarize the selected relative intensities of bands originating from the phenyl and 7-membered rings, the aliphatic chain and the terminal methyl groups. Instead of a complete comparison of all modes of all the molecules, we will focus on a few regions of the Raman and SERS spectra, which can be of a special interest in terms of the adsorption mechanism on the silver
3. Results and discussion UV–Vis absorption spectra of the silver colloid and its mixture with the adsorbate point out what type of SERS mechanism plays a significant role in the adsorption process on a metal colloid. The presence of a single band due to particle plasmon resonance indicates physisorption of a molecule on the sol whereas the appearance of the second peak in the red/near-infrared spectral region suggests a contribution of charge transfer mechanism due to the molecule–metal interaction. UV–Vis spectrum of the silver colloid used in this work exhibits the presence of the plasmon resonance band at ca. 412 nm typical for this sol (Fig. S1B, Supporting Information). After addition of the solution of the drug, absorbance of this band significantly decreases along with blue-shifting of the maximum (Fig. S1C). Broadening of the band indicates the aggregation of the silver particles due to the interaction of the analyte with the silver. Fig. S1C shows an exemplary spectrum for imipramine, however, the spectra of the other TCAs exhibit similar features. In addition, the second plasmon resonance band is observed at ca. 900 nm. This suggests that charge-transfer mechanism may also contribute to surface-enhancement of Raman signal recorded with the use of the 1064 nm laser excitation. For the analytical purpose, we also investigated SERS features of Imi and Des by using a laser excitation at 785 nm [4]. In general, band positions and relative intensities are very similar for both NIR wavelengths of exciting light, indicating a similar mechanism of enhancement of Raman signal.
Fig. 2. Normal (c = 0.2 M) and SERS (c = 2.0 10 4 M) spectra of imipramine (A and B, respectively), desipramine (C and D, respectively) and clomipramine (E and F, respectively).
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A. Jaworska, K. Malek / Journal of Colloid and Interface Science 431 (2014) 117–124 Table 2 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of desipramine in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
411a 495 565 604 685 779 973 1044 1063 1127 1165 1208 1233 1438 1475 1598 2957a 3051a
423 498 565 621 683 774 970 1042 1059 1127 1161 1206 1231 1433 1470 1595 2963 3055
sR6 (29), bR6 (16), bflR62/R7 (9) sR6 (18), sR7 (15), bR6 (8), xN1C/aliph (8) bR7 (33), bR6 (22), xNC/aliph (9) sR6 (42), bR7 (13) bR6 (34), mCC/R7 (19), sR6 (7) sR6 (15), xN2H (8), bR6 (7), mCC/R7 (7) mCC/R7 (32), qCH2/R7 (25) mCC/aliph (32), mCC/R62 (21), mCC/R61 (53), qCH/R61 (12) mNC/aliph (13), bR7 (9), sCH2/R7 (7) qCH/R61 (13), qCH/R62 (9) mCC/R7 (33), bR6 (13), mCC/R62 (7) mNC/R7 (18), qCH/R61 (12), sCH2/R7 (11), mNC/aliph (7) dCH2/aliph (83) dCH2/R7 (80) mCC/R61 (31), mCC/R62 (23) mCH/met (89) mCH/R62 (86)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, btf – butterfly, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs – very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1. a Observed in the Raman spectrum of the solid sample.
Fig. 3. Normal (c = 0.2 M) and SERS (c = 2.0 10 4 M) spectra of amitriptyline (A and B, respectively), nortriptyline (C and D, respectively) and doxepine (E and F, respectively).
Table 1 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of imipramine in the range of 500–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
389 450 498 567 685 973 1044 1063 1127 1207 1232 1436 1473 1597 2971 3062
386 432 500 567 683 972 1042 1061 1127 1206 1229 1431 1468 1597 2963 3057
xNC/R7 (26), sR7 (15), qNC/aliph (7) bNC/aliph (35), sR6 (16), xNC/R7 (7) dCH2/aliph (36), qNC (11) bR7 (23), bR6 (22), xN1C (9) bR6 (32), mCC//R7 (19), sR6 (7) mCC/R7 (32), qCH2/R7 (25) mCC/aliph (38), dCH2/aliph (21), bNC/met (12) mCC/R61 (41), mNC/aliph (8) mNC/aliph (14), bR7 (10), sCH2/R7 (8), mCC/R62 (7) mCC/R7 (33), bR6 (13) mNC/R7 (13), sCH2/aliph (10), mNC/aliph (7), qCH2/aliph (7) bmet (46), dCH2/aliph (42) dCH2/aliph (38), bmet (31) mCC/R62 (35), mCC/R61 (31) mCH/met (89) mCH/R62 (96)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, R6 – 6membered ring, R7 – 7-membered ring, aliph – aliphatic chain, Met – methyl group, vs –very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
nanoparticles. At first glance, the SERS spectra exhibit the presence of all bands observed in the corresponding NR spectra. No shift and significant broadening of SERS bands are observed, thus electromagnetic (long-distance) mechanism of surface enhancement of Raman signal rather plays a leading role in SERS of TCAs. However, the most pronounced feature of all the SERS spectra is a strong enhancement of a band at ca. 680–690 cm 1 attributed to the inplane bending mode of both the flanking phenyl rings (bR6) coupled
Table 3 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of clomipramine in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
370 399 462 507 557 631 692 769 974 1044 1065 1105 1127 1165 1208 1231 1437 1592 2974 3064a
372 399 463 506 556 629 692 770 972 1044 1061 1101 1128 1161 1206 1229 1431 1591 2960 3054
sR6 (36), bR7 (16), bR6 (10) bR7 (30), bR6 (21), mCCl (10) bCN/met (22), sR6 (13) dCH2/aliph (34), sR6 (15), qNC (11), bR7 (7) bR6 (17), bR7 (14), sR7 (10), xNC/aliph (9) sR6 (33), xCCl (21), bR6 (8) bR6 (39), mCC/R7 (18) xCH/R62 (33), bR6 (10) mCC/R7 (32), qCH2/R7 (26) mCC/aliph (36), dCH2/aliph (21), bNC/met (15) mCC/R61 (36), qCH/R61 (17) mCC/R62 (28), b/R6 (7) mCC/R61 (16), qCH/R61 (15), mCC/R62 (11) qCH/R61 (85) mCC/R7 (33), b/R6 (13) mNC/R7 (20), mNC/aliph (11), sCH2/R7 (9) dCH2/aliph (89)
mCC/R62 (41), mCC/R61 (12), mCC/R7 (7) mCH/met (86), mCH/aliph (8) mCH/R62 (97)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs – very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1. a Observed in the Raman spectrum of the solid sample.
with the stretches of the CAC bonds of the 7-membered ring. This mode is a simultaneous in-phase breathing vibration of the dibenzazepine ring. We compare the enhancement of this mode with SERS intensity of the phenyl ring CC stretching vibration (mCCR6) observed at 1600 cm 1 and often denoted as the 8a mode (according to the Wilson’s notation, [22], see PED in Tables 1–6). Since the former is a symmetric vibration, its large enhancement due to the adsorption on the metal surface confirms its origin from electromagnetic rather than from charge-transfer mechanism of the SERS phenomenon [23]. First of all, the enhancement of the 680 cm 1 band gives an evidence for a strong interaction of the conjugated
A. Jaworska, K. Malek / Journal of Colloid and Interface Science 431 (2014) 117–124 Table 4 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of amitriptyline in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
334 534 689 966 1040 1055 1163 1208 1368 1431 1599 1638 2972 3047
336 534 689 968 1038 1053 1163 1208 1366 1427 1597 1638 2963 3042
sR6 (28), bR7 (25) bR6 (28), bR7 (12), sR6 (7), mCC/R7 (7) bR6 (48), mCC/R7 (7) mCC/R7 (29), qCH2/R7 (25), bR61(7) mCC/aliph (56), qCH/R62 (13) mCC/R61 (56), qCH/R61 (12) mCC/R7 (16), mCC/R62 (12), bR6 (11), qCH/R61 (9) mCC/R7 (25), qCH/R62 (12), mCC/R62 (10), bR6 (7) qCH2/aliph (52), mCC/R7 (9) bNC/met (95) mCC/R61 (53), qCH/R61 (13), bR6 (8) mC@C (64), qCH/aliph (9), mCC/aliph (7) mCH/met (94) mCH/R62 (99)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs – very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
Table 5 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of nortriptyline in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
334 534 690 970 1040 1055 1165 1208 1369 1425 1599 1641 2963 3052
336 535 689 968 1038 1057 1163 1206 1370 1427 1597 1641 2963 3044
sR6 (32), bR7 (26) bR6 (27), sR6 (8), mCC/R7 (7), bR7 (7) bR6 (46), mCC/R7 (8) mNC/aliph (29), mNC/met (21), bNC/met (11) mCC/aliph (68), qCH/R62 (15) mCC/R61 (60), qCH/R61 (12) bNC/met (40), mNC/aliph (27) mCC/R7 (30), qCH/R62 (11), mCC/R62 (10), bR6 (7) qCH2/aliph (45), mCC/R7 (10), xCH2/R7 (8) bNC/met (86)
mCC/R62 (54), qCH/R62 (13) mC@C (64), qCH/aliph (9), mCC/aliph (7) mCH/R7 (97) mCH/R62 (95)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs –very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
Table 6 The selected Raman (solution, c = 0.2 M) and SERS (c = 2 10 2 M) bands (in cm 1) of doxepine in the range of 350–3100 cm 1 and their tentative assignments (in %, PED > 7% shown) based on calculations. Raman
SERS
Assignment
343 455 696 838 1041 1050 1165 1221 1369 1461 1604 1638 2960 3060
343 457 696 837 1040 1051 1157 1221 1366 1459 1601 1636 2961 3056
sR6 (32), bR7 (22) qNC/met (23), bR61(10) bR6 (44), mCC/R7 (17), mCC/R7 (8) mNC/met (48), mNC/aliph (20) mCC/aliph (42), mOC/R7 (24), mOC (31), mCC/R62 (24), qCH2/R7 (8) qCH/R62 (45), mCC/R62 (10) mOC (28), qCH/R62 (10), mCC/R7 (10) qCH2/aliph (46), mCC/R7 (14) bCH3 (82)
mCC/R61 (31), mCC/R62 (19) mC@C (63), qCH/aliph (9) mCH/met (96) mCH/R61 (95)
m – stretching, d – scissoring, q – rocking, x – wagging, s – twisting, b – in-plane bending, R6 – 6-membered ring, R7 – 7-membered ring, Met – methyl group, aliph – aliphatic chain, vs –very strong, s – strong, m – medium, w – weak, vw – very weak. Numbering of the functional groups is shown in Fig. 1.
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system of the rings and the silver surface instead of the most common adsorption on the silver nanoparticles via the p-electron system of the aromatic rings [24–26]. In addition, weakening of the 8a mode and the presence of very weak bands originating from the stretching vibration of the aromatic CH bonds (about 3055 cm 1, cf. Figs. 2 and 3) confirm that the phenyl rings are not oriented perpendicularly to the metal surface [27]. Thus, we conclude that the conjugated rings are tilted with the respect to the metal adopting an edge-on geometry. The detailed examination of the SERS spectra of the drugs containing the nitrogen atom in the 7-membered ring reveals that their SERS features differ even due to small structural modifications such as substitution of the methyl group by the hydrogen atom in imipramine and desipramine (Imi, Des, Clo; cf. Figs. 1 and 2). The mentioned-above ratio of bands at 685 and 1600 cm 1 (I683/I1600) in these SERS spectra increases noticeably relative to the corresponding normal Raman bands by a factor of 2–8, confirming the contribution of the dibenzazepine system in the interaction with the silver (Table 7). This ratio in the SERS spectra is similar for imipramine and clomipramine whereas it is a slightly lower for desipramine (see Table 7). Since spatial arrangement of the ring systems in all the antidepressants is similar whereas the I683/I1600 ratio is similar for Imi and Clo containing the chlorine atom in the phenyl ring, we assumed that the loss of the methyl group in the aliphatic chain of desipramine may result in the competition of the other fragment of this antidepressant in the adsorption process and/or a different orientation of its rings with respect to the silver surface. In the case of the latter, we notice variation in the ratio between bands at 683 and 1060 cm 1 (cf. Table 7). According to PED calculations for this group of antidepressants, the 1060 cm 1 band is assigned to the in-phase stretches of the CC bonds of the R61 ring that are non-conjugated to the 7-membered ring (see Fig. 1 for numbering). The I683/I1060 ratio is much lower for desipramine than for imipramine and clomipramine indicating a larger enhancement of the 1060 cm 1 band for Des than for Imi and Clo due to the adsorption on the silver sol. This can suggest that one of the phenyl rings of Des is directed towards the silver nanoparticles whereas Imi and Clo interact through the entire dibenzazepine moiety. Additionally, among the molecules discussed here, the out-of-plane bending modes of the rings observed below 650 cm 1 are enhanced in the SERS spectra of Imi and Des, see Fig. 2 and Tables 1 and 2. They are also present in the SERS spectrum of clomipramine but their intensities are similar to those in the normal Raman spectrum of the solution. This may suggest that the interaction of the Imi and Des aromatic rings with the metal via the p-electron system also appears. Hence, the presence of these modes and the in-plane bending vibrations of the rings indicates a slightly tilted orientation of the dibenzazepine rings of Imi and Des in comparison to Clo. We also examine the change in the integral intensity of the marker bands of the aliphatic chain, i.e. mCC and dCH2 observed at 1042 and 1431 cm 1, respectively, with respect to the 683 cm 1 band (see Table 7). Among the three antidepressants, the SERS spectrum of desipramine exhibits the largest surface enhancement of the mCCaliph mode along with a very low increase in the intensity of the scissoring vibration of the methylene groups. The opposite trend is found for clomipramine. Hence we concluded that the aliphatic chain of Des is oriented almost perpendicularly with respect to the metal whereas it is strongly tilted in clomipramine. In the case of imipramine, the aliphatic chain adopts a intermediate geometry between those proposed for Des and Clo. These orientations of the side chain are also confirmed by the SERS ratio of the 1042 and 1431 cm 1 bands, see Table 7. The second group of the tricyclic antidepressants studied here, i.e. amitriptyline, nortriptyline and doxepine, characterize the presence of the double CC bond attached to the dibenzazepine system,
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Table 7 Relative intensities of selected bands in NR and SERS spectra of imipramine (Imi), desipramine (Des) and clomipramine (Clo).
a b c d
I683/I1600a
I683/I1060b
I689/I1042c
I689/I1431d
I1042/I1431
Imi
NR SERS
0.5 2.2
2.9 11.5
1.6 5.8
7.0 3.4
4.4 0.6
Des
NR SERS
0.7 1.4
2.6 7.8
1.2 4.0
5.7 5.0
4.7 1.2
Clo
NR SERS
0.3 1.9
4.2 11.9
3.1 8.8
2.4 2.0
0.8 0.2
683 cm 1: bR6/mCCR7, 1600 cm 1060 cm 1: mCCR61. 1042 cm 1: mCCaliph. 1431 cm 1: dCH2.
1
: mCCR6.
Table 8 Relative intensities of bands in NR and SERS spectra of amitriptyline (Ami), nortriptyline (Nor) and doxepine (Dox).
a b c d
I690/I1600a
I690/I1640b
I690/I1042c
I690/I1369d
I1042/I1369
Ami
NR SERS
0.6 4.7
0.4 1.9
2.0 6.1
2.9 8.5
1.5 1.4
Nor
NR SERS
0.5 3.1
0.3 1.2
2.0 4.5
2.3 3.8
1.2 0.8
Dox
NR SERS
0.6 3.5
0.4 2.0
3.7 5.5
1.7 4.0
0.5 0.7
690 cm 1: bR6; 1600 cm 1640 cm 1: mC@C. 1042 cm 1: mCCaliph. 1369 cm 1: qCH2/aliph.
1
: mCCR6.
Fig. 4. The proposed model of the adsorption mechanism of the antidepressants (colours of atoms correspond to: gray – carbon, white – hydrogen, blue – nitrogen, green – chlorine, red – oxygen). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cf. Fig. 1. The detailed assignment of Raman and SERS bands is collected in Tables 4–6. Similarly to the group discussed above, the SERS spectra of those antidepressants are similar to the NR spectra in means of bands positions and they also exhibit the enormous intensification of a band at 689 and 696 cm 1 for Ami/Nor and Dox, respectively (Fig. 3). First of all, the I690/I1600 ratio increases 6–8 times in the SERS spectra in comparison to the Raman spectra of the solutions like for the previous group of the molecules, confirming that the dibenzazepine moiety is a primary site of the
interaction with the surface. Interestingly, this ratio is similar for SERS of nortriptyline and doxepine, despite the presence of the oxygen atom in the 7-membered ring of Dox, but the largest value of the ratio is found for amitryptiline. Thus, nortryptiline and doxepine adopt a less tilted orientation of the ring system in comparison to amitryptiline since the 8a mode of the phenyl ring is more enhanced than the bending motion of the three rings together. Moreover, according to our PED calculations, the out-of-plane bending vibration of the phenyl rings conjugated with the in-plane
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bending mode of the R7 ring is manifested by the presence of a weak band at ca. 340 cm 1 (Tables 4–6), which becomes a medium-intensity band in the SERS spectra of all the molecules. Therefore, one can assume that, for this group of the antidepressants, the molecules are oriented in such a way that the benzene rings are preponderantly tilted with respect to the surface. Along with decreasing the intensity of the 8a mode of the phenyl rings due to adsorption on the silver nanoparticles, we also observed lowering intensity of the stretching vibration of the C@C bond (at 1640 cm 1) comparing to the corresponding normal Raman spectra. The intensity ratio of these bands (I690/I1640) in the NR spectra is similar for all molecules indicating significantly larger intensity of the C@C stretching mode than the breathing vibration of the dibenzazepine group. After adsorption on the metal, the latter has an intensity twice as large as the mC@C band in the spectra of Ami and Dox and slightly larger for Nor (cf. Table 8). The molecular structures of the antidepressants studied here rather exclude a simultaneous perpendicular orientation of the dibenzazepine moiety and the C@C bond with respect to the surface (cf. Fig. 4). Thus the ratio between the 690 and 1640 cm 1 bands can indicate to what extend both functional groups are directed perpendicularly to the metal. This ratio is similar for Ami and Dox (ca. 2) whereas it decreases to 1.2 for nortriptyline indicating that the double CC bond is reoriented from a tilted to an upright geometry, respectively. An analysis of the SERS bands attributed to vibrations of the aliphatic chain, mCC (1042 cm 1) and qCH2 (1369 cm 1), exhibits a similar enhancement of the mCC mode with respect to the 690 cm 1 band for all the antidepressants whereas a 2-fold decrease of the I690/I1369 ratio is found for Nor and Dox in comparison to the ratio values calculated for amitriptyline. In turn, the relative intensities of the 1042 and 1369 cm 1 bands clearly indicate an orientation of the side on the silver nanoparticles (see Table 8). Since the I1042/I1369 ratio is similar in the NR and SERS spectra of Ami and Dox, the aliphatic chain is likely oriented in a way similar to the DFT-optimized structures (cf. Fig. 4 and Table 8). In turn, the SERS intensity of the dCH2 mode is higher than the intensity of the mCC vibration for Nor, suggesting that the aliphatic chain of Nor is more tilted to the surface than for Ami and Dox. The reason for that could be the absence of one methyl group in Nor and in consequence a stronger interaction of the lone electron pair of the terminal N atom than in the other molecules. However, the opposite effect is observed for desipramine as discussed above and this clearly results from the different orientations of the dibenzazepine moiety on the silver surface. Summarizing the proposed adsorption models for all antidepressants studied here are illustrated in Fig. 4.
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identified the most favorable location. However, all of them suggest that the tricyclic ring adopts a tilted orientation with respect to the hydrophilic pocket of the serotonin transporter similarly to the TCAs interaction with the metal surface. The possible rotational reorientation of 3-substituted antidepressants like clomipramine from the symmetric docking of imipramine in hSERT could be in the agreement with our SERS spectra that revealed a less tilted orientation of the tricyclic moiety of Clo. A similar orientation of the ring system of nortriptyline was identified in its SERS spectrum that is congruent with the fact that Nor is wedged between transmembrane helices of the dopamine transporter [8]. No detailed data on locations of amitriptyline and doxepin within a transporter have been reported so far in the literature, thus a direct comparison between the SERS model of the adsorption on the metal surface and pharmacological action of the two antidepressants is limited. On the other hand, the proposed models of the surface adsorption for Ami and Dox (Fig. 4) suggest a greater preferential interaction between the p-electron system of the azepine ring and silver nanoparticles in comparison to that observed for the ring system of nortriptyline. Moreover, SERS features of Ami and Dox can implicate that the presence of the oxygen atom in the azepine ring enforces slightly different strength and/or binding fashion with the metallic surface through this moiety. It is also worth mentioning that the binding affinity studies of TCAs have suggested a contribution of chloride ions into the stabilization of their docking pose for a given receptor conformation and they have assumed the presence of one chloride ion in the close vicinity of the antidepressant molecule [2,9]. In turn, in our SERS investigations, we observed weakening of the adsorbate–metal interactions when an excess of chloride ions was added to the mixture of antidepressants and silver sol, taking into account that the TCA:Cl molar ratio of 1:1 is already introduced to the solution. Our results demonstrate an analogy between the molecular structure and orientation of the investigated drugs when interacting with the colloidal silver nanoparticles and their orientation within the central substrate site of the transporters. This suggests, despite the simplicity of the model, that SERS can serve as a probing and complimentary method for experimental pharmacological studies and molecular dynamics simulations. Although, it should be also pointed out that surface-enhanced Raman spectroscopy does not allow identifying the specific amino acid residues as binding sites in the receptors and the type of interaction like p–p stacking, hydrogen bonding or water-mediated salt bridge, but this may rather mimic a docking pose of a ligand within a receptor. Appendix A. Supplementary material
4. Conclusions In this work, we present the orientation of the six tricyclic antidepressants on the silver nanoparticles determined by surface-enhanced Raman spectroscopy. We identified the primary adsorption site on the silver nanoparticles via the p-electron system of the ring moiety and the methyl-aminopropyl side chain. The orientation of the dibenzazepine ring slightly differs between the molecules pointing an effect of the complex structure of the molecules on the subtle changes in their surface behavior. These changes in the orientation of the ring systems are consistent with the interactions of TCAs within the central substrate site of the serotonin and leucine transporters in that the ring system is lying almost perpendicularly in the membrane plane. The alkylamine side chain is oriented similarly to the ring, however, according to molecular dynamics simulations its spatial arrangement varies for the particular antidepressant [2]. Several works cited in Section 1 [2,6,8,9,28] suggest that the position of the particular TCA is flexible in the binding site and in some cases it is difficult to
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