An impact of the ring substitution in nicorandil on its adsorption on silver nanoparticles. Surface-enhanced Raman spectroscopy studies.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

An impact of the ring substitution in nicorandil on its adsorption on silver nanoparticles. Surface-enhanced Raman spectroscopy studies q Aleksandra Jaworska a,b, Kamilla Malek a,b,⇑, Katarzyna M. Marzec b, Malgorzata Baranska a,b a b

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland

h i g h l i g h t s

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

Surface-enhanced Raman spectra of

nicorandil and 1-methylnicorandil are studied. Substituents in the ring nitrogen atom lead to changes in the orientation of the silver. Similarities and differences in SERS profile of each pyridinium species are discussed in detail.

a r t i c l e

i n f o

Article history: Received 8 November 2013 Received in revised form 13 March 2014 Accepted 22 March 2014 Available online xxxx Keywords: Nicorandil 1-Methylnicorandil Raman SERS Ag colloid DFT

a b s t r a c t The substituent effect on structure and surface activity of biologically active nicorandil was investigated by means of surface-enhanced Raman spectroscopy (SERS). Vibrational characterization was a basis for investigation of the adsorption profile of nicorandil and 1-methylnicorandil on silver nanoparticles. An assignment of SERS bands was performed by the comparison of the Raman spectra of both compounds in the solid state and in solutions, complemented by DFT calculations. Even though the nitro group was found to be the most attractive to the silver surface, the strong impact of the methyl substituent changed this preferable adsorption mechanism in 1-methylnicorandil. Protonation of the nitrogen atom in the pyridinium ring was also found to have an impact on absorption mechanism. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Surface-enhanced Raman spectroscopy (SERS) is a useful physico-chemical technique employed for determination of the orientation of molecules adsorbed on metal surfaces such as q Selected paper presented at XIIth International Conference on Molecular spectroscopy, Kraków – Białka Tatrzan´ska, Poland, September 8–12, 2013. ⇑ Corresponding author at: Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland. Tel.: +48 12 663 2064. E-mail address: [email protected] (K. Malek).

colloids, electrodes and highly-ordered nanostructures [1]. Although SERS spectroscopy has been widely applied in studies on various molecules, the mechanism leading to the surface enhancement has not been yet completely understood. Three possible contributions to the enhancement factor have been identified up to now: (i) the surface plasmons resonance (SPR) in metal nanoparticles called electromagnetic effect (EM), (ii) a charge-transfer resonance (CT) involving a shift of electrons between the molecule and the conduction band of the metal, and (iii) resonance of the molecule itself [2].

http://dx.doi.org/10.1016/j.saa.2014.03.104 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: A. Jaworska et al., An impact of the ring substitution in nicorandil on its adsorption on silver nanoparticles. Surfaceenhanced Raman spectroscopy studies, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/ j.saa.2014.03.104

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A. Jaworska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx

The nature of the metal interface strongly influences the way of adsorption of molecules on the metal surface. As the surface of metal nanostructures, such as silver and gold colloids, is negatively charged, the positively charged adsorbates will demonstrate a high affinity to such substrates [1]. On the other hand, molecules’ structure and properties also affect this process. For example, the presence of oxygen, nitrogen or sulphur atoms induces strong metal–adsorbate interactions, whereas highly nonpolar molecules such as polycyclic aromatic hydrocarbons are reluctant to adsorb to the charged interface [1]. In addition, the affinity of the adsorbate toward the metal can be enhanced by modifying the chemistry of the interface, e.g. by using halide anions (most frequently chloride) due to the formation of Ag+ACl ligand surface complexes or due to an increase of the local electromagnetic field by aggregation of metal colloid particles [3,4]. Moreover, SERS is a substituent-sensitive technique indicating how adsorption mechanism varies when the most favourable SERS active sites are blocked by a substituent. For instance, the substitution of the benzene hydrogen atoms by the cyano group changes the orientation of the phenyl ring with respect to the gold surface from the parallel to the perpendicular position [5]. From a biological/pharmacological point of view, SERS can provide a first hint what kind of drug-receptor interactions may play a significant role in mechanism of action on the molecular level. For example, SERS studies suggested that 9-aminoacridine in the amino monomeric form may interact with the enzyme through the anionic bond with a negatively charged amino acid or by a ring stacking interaction with an aromatic residue placed in the catalytic site of trypsin-like protease guanidinobenzoatase [6]. Similarly, interactions of antitumoral drug emodin with human serum albumin (HSA) have been evaluated from SERS spectra showing two potential binding sites of the drug through the IIA and IIIA hydrophobic pockets of HSA [7]. In this work the comparison of the vibrational profile and surface activity of nicorandil (N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide, NICO, Fig. 1A) and 1-methylnicorandil (1-methyl-3-N0 -[(nitrooxy)ethyl]-nicotinamide chloride, MNIC+, Fig. 1C) is demonstrated. This is also a continuation of our previous SERS studies on pyridinium salts [4,8]. Nicorandil, a hybrid ATP-dependent potassium (KATP) channel opener, is used clinically for the treatment of angina pectoris, and so far it is the only anti-anginal agent to have significantly improved outcome in patients with a stable angina [9 and therein]. The cardioprotective action of nicorandil in ischemic hearts have received much attention since this compound can improve the recovery of post-ischemic contractile dysfunction and reduce infarct size in several animal models as well as in humans [9]. In turn, 1-methylnicorandil belongs to 3-substituted pyridinium salts, which have been shown to exhibit cytotoxic properties against murine leukemia L1210 [10]. However, a mechanism of pharmacological action of these pyridinium salts has not been yet recognised, therefore our SERS studies on both molecules can provide a first insight into their interaction with a surface, and consequently into drug action. To our best knowledge, only FTIR and Raman spectra of nicorandil in the solid state have been reported so far [11].

Since the nitrogen atom of nicorandil can be protonated, we investigated Raman and SERS spectra of NICO at three different pHs Next, this enabled us to perform comparative studies of adsorption features of both molecules with the substitution in the ring N atom, the most potential SERS-active site, by the single atom and the bulk group. In SERS studies, we used silver colloid and a Vis-excitation line (532 nm). The experimental studies of nicorandil and its methyl derivative were complemented by quantum-chemical calculations at the DFT(B3LYP)/6-311++G(d,p) level of theory. Experimental Chemicals Nicorandil and 1-methylnicorandil were synthesized in the Technical University of Lodz, Institute of Applied Radiation Chemistry, Poland. Purity of compounds was confirmed by elemental analysis, 1H NMR spectroscopy and LC/MS. All other chemicals were purchased from Sigma, Germany, and were of analytical grade. 1 and 1 102 M aqueous solutions of NICO and MNIC+ were prepared by using 4-fold distilled water. pH of aqueous solutions of NICO and MNIC+ after dissolving was 4 and 5, respectively. NICO solutions with pH of 1 and 11 were prepared by addition of 1 M HCl and 1 M NaOH, respectively. Silver colloid was synthesized according to the procedure described previously [12], in which Ag ions are reduced in the alkaline solution of hydroxylamine. UV–Vis spectra of several colloid batches showed the presence of an absorption band at ca. 409 nm, typical for this silver colloid. To accomplish SERS measurements, 10 lL of the adsorbate solution was mixed with 500 lL of colloid previously activated with 20 lL of 0.5 M KCl solution. Thus, the final concentration of the analytes in the mixture was 1.9 104 M. Instrumentation Raman and SERS spectra were recorded on a Witec Alpha 300 Raman microspectrometer equipped with a confocal microscope, a 532 nm excitation line and a CCD detector. The spectral resolution was 3 cm1 and 8 scans were collected for all measurements with an integration time of 10 s (Raman of solids and solutions and SERS). A few milligrams of solids and 200 lL of solutions were placed on metal discs and in a 96-well plate, respectively. SERS spectra were collected three times by using three batches of colloid and were reproducible. Here, we present average spectra for each adsorbate. Low signal to noise ratio in SERS spectra was observed for solutions with concentration below 1.9 104 M. Electronic absorption spectra were recorded with a UV–Vis–NIR Perkin Elmer spectrophotometer (model Lambda 35) in the range of 200–1100 nm with a resolution of 1 nm. UV–Vis measurements were carried out placing the solution in quartz cuvettes of 1 cm thickness. Computational methods Density Functional Theory (DFT) calculations were carried out using the Gaussian 09 program package [13]. To fully characterize

Fig. 1. Chemical structures of: a neutral form of nicorandil (A), a protonated form of nicorandil (B) and 1-methylnicorandil (C).



the experimental SERS spectra for studied compounds, an extensive series of theoretical calculations on the various possible conformers of nicorandil and 1-methylnicorandil were carried out. This provided relevant structural and energetic data as well as vibrational information crucial for the analysis of the experimental SERS spectra. The geometries of the most stable 16 conformers of nicorandil, protonated nicorandil, and 1-methylnicorandil were fully optimized at the B3LYP level of theory with the 6311++G(d,p) basis set. Harmonic frequencies and Raman activities were computed for all of those conformers. The theoretical wavenumbers were scaled down by the appropriate scaling factors (0.978 and 0.960 for the 0– 2000 and 2000–4000 cm1 regions, respectively [14]). To provide an unambiguous assignment of the calculated Raman spectra, normal coordinate analysis was performed using a set of internal coordinates defined as suggested by Pulay and Fogarasi [15,16]. The GAR2PED program was used for calculation of potential energy distribution (PED) of normal modes in terms of natural internal coordinates [17].

Results and discussion Even small differences in molecular structure and charge distribution of the studied compounds can induce spectral changes and may involve differences in the orientation of the molecule on the metal surface. In the case of 1-methylnicorandil, the presence of methyl group provokes the transition of nitrogen to a quaternary oxidation state and, as a result, the molecule exists in a cationic form. Thus, nicorandil is a neutral molecule, whereas its N-methyl derivative forms a chloride salt (Fig. 1). Molecular structure of nicorandil in crystals is stabilized by intramolecular van der Waals interactions between the carbonyl O atom and the nitro groups as well as by intermolecular H-bonding [18]. In turn, crystallographic structure of MNIC+ has not been yet reported in the literature, however one can expect similar interactions between functional groups in the side chain of the molecule. Theoretical Raman spectra of optimized structures for all possible conformers of NICO, NICOH+ and MNIC+ in vacuo were compared with experimental spectra. However, one must pay attention that appearance of relevant intermolecular interactions between the closely located molecules of the solute can favour conformers with higher energies than those predicted as the most energetically preferable structures [19]. Therefore, in our selection of theoretical spectrum suitable for further PED analysis we considered the most stable conformers as well as less stable structures in which interactions between the O and H atoms in the side chain can simulate molecular structure in the solid. Molecular structure of the conformers are depicted in Figs. S1 and S2 along with theoretical Raman spectra (in Supplementary Materials). The comparison between theoretical and experimental Raman spectra of 1-methylnicorandil suggests that spectra of the two most stable forms of MNIC+ (MNIC+ 1 and MNIC+ 2, Fig. S2) are found in a good agreement with FT-Raman spectrum. In these conformers the ring and the side chain are coplanar. In contrast to this compound, the energetically preferred structure of the NICO molecule shows an almost perpendicular arrangement of the side chain with respect to the ring. However, a better agreement between the computed and experimental Raman spectra is found for NICO 3 then for the most stable conformer - NICO1 (see Fig. S1). The most important difference in theoretical spectra between NICO and its protonated form is blue-shift of bands in the spectral region of 2900–3100 cm1. Such a behaviour explains the shift in experimental spectra of NICO solutions as it will be discussed below. It is well known that differences between theoretical spectra for optimized molecules in gas phase and experimental

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spectra of compounds in condensed phases are expected. However, these differences are not significant here, thus choosing the mentioned-above structures for calculations of PED allowed us for the assignment of the most pronounced spectral features found in Raman spectra of nicorandil and 1-methylnicorandil. In addition, the bands assignment discussed below agrees well with previous results for similar compounds [4,8,11].’’ Features of Raman spectra Both, nicorandil (protonated and neutral forms) and 1-methylnicorandil, belong to the same C1 symmetry group and hence all the vibrational modes are Raman active. The assignment of vibrations to Raman bands was carried out by the comparison of Raman spectra of the solids with the corresponding calculated ones. Theoretical Raman spectra of NICO, NICOH+, and MNIC+ are displayed in Figs. S1 and S2 in Electronic Supporting Information. Normal Raman spectra of NICO and MNIC+ in the solid state recorded with the use of a 532 nm excitation line show several well-resolved bands, which correspond to spectral differences between the molecules (Figs. 2A and 3). Tables 1 and 2 summarise the band assignment for NICO and MNIC+, respectively. As it was previously reported the theoretical spectra mainly allowed for the assignment of bands in the solid state, however the use of this assignment is also helpful in understanding spectral information for molecules in solution as well as surface enhancement of Raman signal SERS spectra [4,8,20,21] Since the aim of our studies is the elucidation of the SERS profile recorded for solutions, we focus firstly the discussion on spectral features of normal Raman spectra of concentrated aqueous solutions of the studied compounds. After dissolving solids in water (1 M solutions), most of Raman bands occurring in the spectra of solids for both molecules disappear exhibiting in general a similar Raman profile (see Figs. 2A and 3). Since, only molecular structure of nicorandil is pH-sensitive (MNIC+ is already in a cationic form), Raman spectra for solutions at pH of 1, 4, and 11 were recorded. pH of 4 is characteristic for the NICO solution after dissolving. Since the pK value for this compound has not been established up to now, we assumed that the neutral form of nicorandil (Fig. 1A) exists in alkaline solution (here at pH of 11) whereas protonation of the ring nitrogen atom (Fig. 1B) appears in strongly acidic environment (pH around 1). This is also supported by the pK range of 2–4 typical for other pyridinium salts [8]. Our Raman spectra of solutions show the presence of a few bands only. The strongest band seen in all Raman spectra of NICO is observed at 1051 cm1, with a small shoulder at 1037 cm1 appearing at pH of 4 and 11. The former is assigned to a coupled stretches of the CC and CN bonds (mode 12 according to the Wilson’s notation) whereas the latter represents the ring breathing vibration (mode 1). Interestingly, the relative intensity of those two bands is reversed in the comparison to the counterparts in the Raman spectrum of the solid state. A similar finding was observed for the pyridinium halides (PyHX) studied by Raman spectroscopy, for which the increase in intensity of the mode 12 was explained by formation of very strong H bonding between molecules of water and the pyridinium ring [22]. However, the major spectral differences between the spectra of the solutions at three different pH are observed in the region of 1500–1700 cm1. In the Raman spectrum of the solid nicorandil, two prominent bands at 1636 and 1596 cm1 appear in this region and they are assigned to the asymmetric stretching vibration of the nitro group (masNO2) and the 8a mode of the ring, respectively. Next, the spectrum of the solution at pH of 1 exhibits the presence of a medium-intensity band at 1644 cm1 only that could be treated as a counterpart of the 1636 cm1 band seen for the solid. However, one should consider that the protonated molecules of NICO present in this solution are now the 1,3-disubstituted molecules instead of


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Fig. 2. A comparison of normal Raman spectra of the solid and 1 M solutions at pH of 1, 4, and 11 (A) and SERS spectra of 1.9 104 M solutions at pH of 1, 4, and 11 (B) of nicorandil.

Fig. 3. A comparison of normal Raman spectra (C) of the solid of 1 M solution (B) and SERS spectrum of 1.9 104 M solution (A) of 1-methylnicorandil.

the mono-substituted ring. Therefore, we propose to assign the 1644 cm1 band to a coupled 8b (PED: 51%) and NAH rocking modes (PED: 24%) according to DFT and PED calculations for the protonated form of NICO. This indicates that this band is a marker of the protonated form of the pyridinium ring. A similar band was found and assigned to the same set of vibrations in Raman spectrum of nicotinamide at pH of 2 [8]. Next, by increasing pH of the NICO solution to 4, the second band appears at 1602 cm1 (8a) in the Raman spectrum whereas in the strongly alkaline environment the 1644 cm1 band disappears along with increasing the intensity of the 8a band, c.f. Fig. 2A. The latter is accompanied by the aromatic CH stretching vibrations at 3072 cm1. This comparison suggests that the protonated and neutral forms of NICO are present at pH of 1 and 11, respectively, while both forms likely exist at pH of 4. According to our calculations, other Raman bands found in the spectra of all solutions at 630, 840–850, and 1195– 1205 cm1 are attributed to the pyridinium ring modes whereas bands at 1390 and 2983 cm1 originate from vibrations of the methylene groups of the side chain (see Table 1 for details). The Raman spectrum of the aqueous solution of 1-methylnicorandil also exhibits the presence of a few bands only (Fig. 3). It is dominated by a peak at 1039 cm1 (mode 1) with a shoulder at 1051 cm1 (a coupled COANO2 and CAC stretches of the side chain). Similarly to spectral changes observed for NICO, the intensity of the latter arises significantly due to interaction with water molecule. In addition, a broad band centred at 1645 cm1 with a shoulder at 1603 cm1 (8b and 8a, respectively) is observed in the region typical for vibrations the ring (see Table 2). The broadening of this band results from a contribution of the bending vibrations of water molecules. Other bands of low intensity (1286, 1335 and 1388 cm1) originate from the stretching and in-plane bending modes of the ring and side chain moieties while the high-wavenumber region obscured by a



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Fig. 4. UV–Vis absorption spectra of Ag colloid (black trace), Ag colloid activated with KCl (red trace), activated colloid mixed with solution of the adsorbates (green trace) and solutions (blue trace) of nicorandil (pH = 4) (A) and 1-methylnicorandil (pH = 5) (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1 Theoretical (DFT) and experimental bands (cm1) in the Raman spectra of nicorandil in the solid state (solid), in the aqueous solution (aq) and SERS spectra (SERS), with the assignmenta,b based on the DFT(B3LYP)/6-311++G(d,p) calculations and potential energy distribution (PED, %, contributions lower than 10% omitted). Nicorandil DFT 468 482 517 620 697 826 847 885 919 994 1021 1037 1111 1150 1200 1225 1267 1296 1370 1434 1474 1596 1625c 1690 2940 2994 3059 3074

Solid

aq pH = 1

aq pH = 11

SERS pH = 1

SERS pH = 4

SERS pH = 11 472

543 627 693 827 858 890 906 1039 1059 1118 1141 1175 1207 1284 1330 1368 1426

553 626 813 842 875

1051

1195

1389 1468

1596

637 695 829

692 821

692 832

910 993 1035

904 993 1035

910 993 1035

1205

1213

1153 1208

1111 1143 1205

1326

1321

1311

850

1037 1051

1395 1599

1644 1636 2956 2975 3075 3099

1341 1377 1427

2983

1590 1639

1590

1590

1634 2954

1632 2956

3091

3091

2976 3072

PED (contributions > 10%) bCCO(14), dCH2(12), sCN(11) sCN(35), xNH(30) dCH2(23), bCCO(13), xCC/ali(10), sR6(11) bR6(86) bR6(38), mON(13) xCH/R6(51), sR6(20), xout/CN/ali(12), xCC/chain(12) qNO(37), mON(33), bNO2(18) qCH2(22), mNC/ali(15), mCC/ali(12), mCO(9) mCO/NO2(19), mCC/ali(13), bCNO(12) xCH/R6(85) bR6(50), mCC/R6(19); (mode 1) mCC/R6(35), qCH2(11), mNC/R6(10); (mode 12) qCH/R6(46), mCC/R6(15), mNC/R6(11) mNC/ali(46) qCH/R6(39), mNC/R6(27), mCC/R6(11) sCH2(83) mNC/R6(38), mCC/R6(37) sCH2(43), mNO2 sym(19) xCH2(70) dCH2(91) qCH/R6(48), mNC/R6(8), mCC/R6(14) mCC/R6(44), mNC/R6(15); (mode 8a) mCC/R6(48), qCH/R6(20), mNC/R6(14); (mode 8b) and/or mNO2/asym(89) mNO2 asym (88) mCH2 sym(99) mCH2 asym(99) mCH/R6 sym(99) mCH/R6 asym(98)

a

m – stretching; b – in-plane bending, q – rocking; x – wagging; d – scissoring; c – out-of plane bending; ali – atom(s) from the chain; R6 – ring.

b

Internal coordinates are defined in Table S1 (Electronic Supporting Materials). An assignment based on theoretical spectrum of NICOH+.

c


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Table 2 Theoretical (DFT) and experimental bands (cm1) in the Raman spectra of N-methylnicorandil in the solid state (solid), in the aqueous solution (aq) and SERS spectra (SERS), assignmenta,b based on the DFT(B3LYP)/6-311++G(d,p) calculations and potential energy distribution (PED, %, contributions lower than 10% omitted). N-methylnicorandil DFT

Solid

aq

685 777 1023 1029 1187 1227 1244 1278 1364 1588 1632 1714 2998 3030

688 759 1033

696 765 1039 1051

1206 1241 1280 1322 1380 1593 1630 1664 2965 2978

SERS 701 1038 1216

1286 1335 1388 1601 1645

1598 1637 2960

2977

PED (contributions > 10%)

mON(23), bR6(16), bCON(13) qCN/ali(25), mCN/CH3(13), xCNC(12) bR6(32), mCC/ali(10), mCO/NO2(9), mCC/R6(24) (mode 1) mCO/NO2(29), mCC/ali(24), bR6(12) mCN/CH3(25), bR6(17), mCN/ali(19) sCH2(68), sCNC/ali(19) sCNC(19), bHNC/ali(17), mCC/ali/R6(10), qCN/ali(8) mNOsym(51), xCH2/ali(19), sCH2/ali(9), qNO(7) qCNC/ali(51), xCH2(28) mCC/R6(42), qCH/R6(14), mCN/R6(19) (mode 8a) mCC/R6(46), mCN/R6(12), qCH/R6(16) (mode 8b) mNO/asym(88), bONO(7) mCH2/asym(99) mCH3/asym(99)

a

m – stretching; b – in-plane bending, q – rocking; x – wagging; d – scissoring; c – out-of plane bending; ali – atom(s) from the chain; R6 – ring.

b

Internal coordinates are defined in Table S1 (Electronic Supporting Materials).

strong and broad band of the water OH stretches reveals only the presence of a band at 2977 cm1 assigned to the symmetric stretching mode of the ring CH3 group (see Table 2). Adsorption on Ag colloid. SERS In order to assess the nature of the interactions between silver nanoparticles and adsorbates studied here, electronic absorption spectra were recorded for the colloid and its mixture with NICO and MNIC+ (Fig. 4). It can be easily noticed that electronic absorption spectra of the activated colloid after addition of NICO and MNIC+ solutions differ in the 600–1100 nm region, showing the second surface plasmon band in the spectrum of NICO only. This suggests a contribution of a mixture of charge-transfer and surface plasmon resonances in surface Raman enhancement of nicorandil whereas only SPR effect is likely responsible for SERS of 1-methylnicorandil [2]. Taking into consideration the differences in the molecular structures of NICO and MNIC+ as well as the protonated form of nicorandil, adsorption mechanism of both compounds on silver colloid should exhibit different features. Accordingly to our previous studies on SERS of pyridinium salts [4,8], one should expect an adsorption fashion through the p-electron rich groups in the side chain if the ring nitrogen atom is blocked by an atom or a group of atoms, whereas a direct interaction between the free nitrogen atom of the pyridinium ring with the silver should be preferable

in a similar manner to pyridine [23]. Similarly to normal Raman studies, SERS spectra of nicorandil were collected at different pH environment (Fig. 2B). Likewise in the case of Raman spectra of aqueous solutions, there are some significant differences appearing according to the different pH. A couple of bands at 1035 and 993 cm1 appears in each spectrum. As mentioned above, we attributed the strong band at 1035 cm1 to the breathing mode of the pyridinium ring (mode 1), whereas our calculations of Raman spectrum reveal that the shoulder at 993 cm1 originates from the wagging CH motions in the pyridinium ring. It is worth mentioning that a similar band appears in SERS spectrum of nicotinamide at pH = 9 [8] but not in SERS spectra of MNIC+ (Fig. 3), trigonelline [8], and dimethyl derivatives of the pyridinium ion [4]. Since the latter regards the molecules with the methyl group substituted to the pyridinium ring, the appearance of the 993 cm1 band may indicate the presence of the neutral form of the ring interacting with the silver surface. Introducing different forms of nicorandil molecules (at pH of 1 and 11) to the silver colloidal suspension results in significantly different SERS profile in the spectral region above 1100 cm1, c.f. Fig. 2B. A few bands appear in the SERS spectrum for pH of 1, which are assigned, according to our PED calculations, to the vibrations of the protonated form of NICO (c.f. Table 1). The marker band for the protonated form of NICO is SERS-active at 1639 cm1 whereas the 8a mode appears at 1590 cm1 in SERS spectra of each studied solutions and it is shifted by 9 cm1 in the comparison to the nor-

Fig. 5. Proposed models of adsorption mechanism of nicorandil, protonated nicorandil and 1-methylnicorandil on silver nanoparticles.



mal Raman spectrum (see Fig. 2A and B). This shift probably results from the contribution of charge-transfer mechanism into SERS of nicorandil, as mentioned above. However, one can note that the asymmetric stretching mode of the NO2 group appears at 1636 cm1 in the Raman spectrum of the solid sample, thus a contribution of this vibration to the SERS band at 1639 cm1 cannot be unambiguously excluded. Furthermore, this region of the SERS spectrum for pH of 1 is similar to the normal Raman spectrum of the solution at pH of 4, suggesting that some population of the NICOH+ ions undergoes deprotonation due to the contact with the metal surface, and thus both forms of nicorandil exist on the silver sol in these conditions. Removal of labile protons is a well recognised fact in the SERS phenomenon [21]. In addition, the symmetric stretching mode of the nitro group and the AOANA stretch are present in the SERS spectra of all the solutions at ca. 1321 and 695 cm1, respectively, along with the twisting vibrations of the neighbouring >CH2 groups. At pH of 1, a small band at 2976 cm1 additionally appears in the high frequency region [mas(CH2)] with the absence of the CH stretches of the pyridinium ring. These findings implicate that the NO3 group is involved in the interaction with the Ag surface through the p-electron system oriented horizontally whereas polarizability components of the CH2 stretching mode are perpendicularly directed towards the surface. Thus, the lack of the aromatic CH vibrations indicates that the pyridinium ring does not interact vertically with the metal but rather in a bent orientation when the molecule is protonated. In the SERS spectrum recorded for the solution of pH = 11, intensities of a few bands in the region of 1300–1450 cm1 increase in the comparison to the other SERS spectra, see Fig. 2B. The strong doublet at 1341 and 1377 cm1 originates from coupling of the CH2 twisting and symmetric stretching NO2 modes and the CH2 wagging vibration, respectively. Broadening of those bands indicates various orientations of the NICO side chain on the silver surface. This could be associated with shifting of a band at 1639 cm1 observed at pH of 1 to the wavenumber of 1632 cm1. Here, we propose to assign the 1632 cm1 band to the asymmetric stretching vibration of the NO2 group instead of the mixture of the q(NH) and 8b modes, since no protonated molecules are expected to be present in alkaline environment. The appearance of the asymmetric as well as symmetric stretches of the nitro group indicates that this moiety in the molecules of NICO adopts a vertical orientation with respect to the silver nanoparticles. Additionally, enhancement of a band at 3091 cm1 assigned to the m(CHR6) mode supports the fact that the ring is perpendicular to the metal. On contrary to the SERS spectrum at pH of 1, the symmetric instead of asymmetric stretching motions of the methylene groups are SERS-active at pH of 11 (a band at 2956 cm1). Nevertheless this also indicates involving the CH2 groups in the interaction with the Ag colloidal particles. Summarising the SERS behaviour of nicorandil in acidic and alkaline solutions, the molecules change their way of interacting with the silver surface from a bend orientation of the ring with a planar OANO2 group to a vertical orientation, where the ring and the terminal nitro group are co-planar. At pH of 4, the lack of the strong SERS bands at ca. 1320 and 1380 cm1 suggests that the neutral molecules do not dominate in the adsorption process on the silver nanoparticles but their presence may be confirmed by appearing of the mas(NO2) and m(CHR6) bands. 1-methylnicorandil, on the other hand, is present in the solution in a cationic form, but its similarity to the SERS spectrum to the protonated nicorandil at pH of 1 is rather coincidental (Figs. 2B and 3). In the SERS spectrum in Fig. 3A, the strongest band at 1034 cm1 is assigned to the ring breathing vibration (mode 1), and this is neither shifted nor broaden in the comparison to the spectrum of MNIC+ in solution (Fig. 3B). Bands at 1598 and 1637 cm1 assigned to the 8a and 8b modes, respectively, are sig-

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nificantly enhanced in the SERS spectrum. On the contrary to the Raman spectra for the solid and solution, in SERS the symmetric 8a band is more intensified than the asymmetric vibration of the ring C@C bonds. The appearance of SERS strong ring bands attributed to the symmetric modes of the pyridinium ring, i.e. 1 and 8a, is typical for a contribution of electromagnetic mechanism in surface enhancement of the aromatic rings, however the mode 8b indicates also the presence of the CT effect, even though the second surface plasmon band is not clearly observed in electronic absorption spectrum (Fig. 4) [8,22]. Since the stretching vibrations of the ring CH bonds are absent in the SERS spectrum of MNIC+, a vertical orientation of the pyridinium ring is excluded. The high-wavenumber region is dominated by a strong and broad band at 2960 cm1 originating from the asymmetric stretching vibration of the methylene group according to our calculations. This indicates that polarizability components of this mode are oriented perpendicularly to the silver surface. Interestingly, there is no indication that the terminal AOANO2 group interacts with metal particles, since neither symmetric nor asymmetric stretching motions appear due surface enhancement at wavenumbers of ca. 1320 and 1660 cm1, respectively. These bands were attributed to vibrations of the nitro group according to the comparison of the Raman spectrum of the solid with DFT calculations. Among pyridinium salts studied so far in our laboratory, the features of SERS bands observed here are similar to those assigned to the ring modes of trigonelline [8].

Conclusions SERS studies on nicorandil and its methyl derivative show that the substituent attached to the ring nitrogen atom enforces a way of the adsorption on the silver nanoparticles. If there is a single H atom or no substituent like in nicorandil, the p-electron system of the AOANO2 group interacts strongly with the Ag colloidal particles. In addition, the absence of the NH group in the ring of nicorandil leads to re-orientation of the nitro group, whereas steric hindrance in the side chain of nicorandil causes simultaneously a perpendicular orientation of the CH2 groups to the metal. The methyl group in 1-methylnicorandil prevents the adsorption through the nitro moiety and a perpendicular orientation of the pyridinium ring. The pyridine ring of both salts is involved in the interaction with silver nanoparticles, and when the N atom is blocked the ring adopts a tilted orientation in both molecules. Due to removal of the hydrogen atom from the ring, its position becomes perpendicular. The proposed models of adsorption mechanism of the studied here molecules on silver nano particles are illustrated in Fig. 5. The comparison of the SERS behaviour of the pyridinium derivatives studied here and others like nicotinamide, 1-methylpyridinium ion, trigonelline and dimethylpyridines exhibits different adsorption mechanisms, which in consequence may reflect a different action of these potential drugs.

Acknowledgements This work was supported by the European Union under the European Regional Development Fund (grant coordinated by JCET-UJ, POIG.01.01.02-00-069/09). The computational part of this research was supported by PL-Grid Infrastructure and Academic Computer Centre Cyfronet in Krakow (Poland).

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.03.104.


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