- Email: [email protected]
The temperature-dependent single-crystal Raman spectroscopy of a model dipeptide: l -Alanyl-l -alanine
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
The temperature-dependent single-crystal Raman spectroscopy of a model dipeptide: L-Alanyl-L-alanine J.G. Silva a, L.M. Arruda a, G.S. Pinheiro a,b,⇑, C.L. Lima b, F.E.A. Melo a, A.P. Ayala a, J. Mendes Filho a, P.T.C. Freire a a b
Departamento de Física, Universidade Federal do Ceará, C.P. 6030, CEP 60455-760 Fortaleza, CE, Brazil Departamento de Física, Campus Ministro Petrônio Portella, Universidade Federal do Piauí, CEP 64049-550 Teresina, PI, Brazil
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
Temperature dependent polarized
Raman spectra of the L-alanyl-Lalanine. The crystal of L-alanyl-L-alanine undergoes a structural phase transition. The phase transition is observed between 80 and 60 K.
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 4 April 2015 Accepted 7 April 2015 Available online 11 April 2015 Keywords: Raman spectroscopy Infrared spectroscopy Peptide Phase transition
a b s t r a c t A single-crystal of peptide L-alanyl-L-alanine (C6H12N2O3) was studied by Raman spectroscopy at lowtemperature, and a tentative assignment of the normal modes was given. Evidence of a second order structural phase transition was found through Raman spectroscopy between the temperatures of 80 K and 60 K. Group theory considerations suggest that the transition leads the sample from the tetragonal to a monoclinic structure. Additionally, our study suggests that the mechanism for the structural phase transition is governed by the occupation of non-equivalent C1 local symmetry sites by the CH3 molecular groups. Analysis based on group theory suggests L-alanyl-L-alanine presents C2 symmetry at low temperatures. Ó 2015 Elsevier B.V. All rights reserved.
Introduction Peptides are important organic compounds not only because of their ability to form proteins but because of the central role they play in several biological and physiological processes in living organisms [1]. Among the biological functions displayed by peptides are their capabilities as neurotransmitters and hormones. ⇑ Corresponding author at: Departamento de Física, Universidade Federal do Ceará, C.P. 6030, CEP 60455-760 Fortaleza, CE, Brazil. Tel.: +55 8533669906; fax: +55 8533669450. E-mail address: gardenia@fisica.ufc.br (G.S. Pinheiro). http://dx.doi.org/10.1016/j.saa.2015.04.010 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Additionally, they are important for being constituents of several protein chains and taking part in our biological metabolism. As a consequence, peptides have received special attention from researchers of the pharmacological area. Further uses as, for instance, antibiotic and immunological agents are also active fields of study for peptides [2]. As a consequence, the study of peptides has considerably grown in importance in the last few decades. Although its origin is credited to Emil Fischer for his article on the synthesis of the dipeptide glycylglycine in 1901 [3], the subject remained relatively dormant for years due to difficulties the peptide synthesis presented. This difficulty was solved gradually with increasingly efficient
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
245
found in the literature, although some works performed at room temperature were presented [15–18]. The understanding of vibrational and structural properties of peptides is of relevance since they have potential use in medicine. In this work, the vibrational properties of Ala-Ala through Raman spectroscopy at low temperatures are investigated, and an assignment of the vibrational modes is provided. Careful analysis of the behavior of the vibrational modes as a function of temperature is also furnished. Fig. 1. Experimental and calculated powder X-ray diffractogram of L-alanyl-Lalanine crystal.
Experimental procedures
temporary protections for the amino group. After the synthesis of an active hormone, the octapeptide oxytocin, by du Vigneaud et al. in 1954 [4], the variety of synthesized natural occurring peptides and analogs grew along with the demand for them and the study of their properties. The study of peptides is varied in scope and methods [5–9]. Vibrational spectroscopy has become an important tool for the investigation of protein and peptide science and as such, several studies on vibrational properties of peptides have been conducted [10–14]. L-Alanyl-L-alanine (Ala-Ala) is one of the simplest peptides but any study of its vibrational properties at low temperatures are
Single-crystal samples of Ala-Ala (C6H12N2O3) were grown by the vapor-diffusion technique at controlled temperature (278 K). The crystals obtained were thin platelets shape and colorless. The crystal structure of the samples obtained was confirmed through an X-ray powder diffraction experiment using a D8 Advance (Bruker) diffractometer using Cu Ka radiation operating at 40 kV, 40 mA. The Raman spectra of Ala-Ala were obtained using a T64000 Jobin Yvon spectrometer equipped with a liquid N2-cooled charged coupled device detection system and a 514.5 nm argon ion laser. The slit of the spectrometer allowed a resolution of 2 cm1. In order to obtain low temperatures, the sample was placed in a helium flux cryostat (model DE202S, from Air Products and Chemicals), and the temperature was monitored in the interval
Fig. 2. (a) Molecular structure of Ala-Ala; (b) unit cell of Ala-Ala crystal.
(a)
(b)
Fig. 3. Raman spectra of Ala-Ala crystal in the 50–600 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
246
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
20–300 K by a digital temperature controller unit having a stability of 0.1 K.
Scattering geometry
Results and discussion Ala-Ala crystallizes in the tetragonal structure belonging to the I4 (C54) space group [19]. The lattice parameters are a = b = 17.985 Å and c = 5.154 Å, and contains eight formulas per unit cell. Fig. 1 shows the Ala-Ala X-ray diffraction pattern obtained at room temperature, refined by Rietveld method with R-factors Rwp = 10.3% and S = 1.2 for the goodness of fit. The data obtained is good agreement with the pattern previously published [19]. The molecular structure and a view of the unit cell are shown in Fig. 2(a) and (b). The structures were visualized by VESTA [20]. The molecular packaging of Ala-Ala is formed by eight hydrogen bonds between the nitrogen of one molecule with carboxylate oxygens from four different molecules. Factor group analysis shows that the 273 optical modes predicted decompose into the irreducible representations as C = 68A + 69B + 68E. All representations are Raman active and the A and E representations are also infrared active. The axes of the crystal were defined according to the following convention: the z-axis was that perpendicular to the plane of the platelet, the y-axis was defined as the axis coincident with the longest dimension of the crystal and the x-axis was defined perpendicular to the y- and z-axes. The Raman spectra were taken for
two
scattering
Table 1 Vibrational wavenumbers (cm1) and tentative assignment for the Ala-Ala from spectra recorded at room temperature.
geometries:
ðx yÞðz; zÞðx yÞ
and
ðx yÞðx þ y; zÞðx yÞ, which provide data on the modes with A and E symmetries, respectively. In this work, the usual Porto’s notation [21] was used for describing the scattering geometries. Because of the platelet form of the crystals obtained, the z(xy)z geometry was not available, and the phonons of the B irreducible representation are not presented in this work. Fig. 3 shows the Raman spectra of Ala-Ala for several temperatures in the spectral range 50–600 cm1, for (a) the ðx yÞðz; zÞðx yÞ and (b) the ðx yÞðx þ y; zÞðx yÞ scattering geometries. All the spectra presented in this work were taken in the cooling cycle. This region is characterized by the external modes of the crystal and by internal modes of low-energy. These bands provide important insights about the structural stability of the material [22–24]. The band at 153 cm1 is associated with 1 the torsion of CO is 2 unit, s(CO2 ), and the band at 242 cm assigned as the torsion of CH3, s(CH3). A band of great importance due to its relationship with the hydrogen bond is torsion of NH+3, s(NH+3), which is observed at 473 cm1 in the Raman spectrum. Table 1 presents the tentative assignment of the bands observed in the Raman spectra of Ala-Ala at room temperature for the two scattering geometries. Under cooling, the Raman spectra in Fig. 3 show great changes in both scattering geometries between 60 and 80 K. An increase in the number of the modes in the region 50–200 cm1 is observed indicating a change of symmetry of the crystal. Furthermore, in the region 200–600 cm1, the splitting of bands associated with both the torsion of CH3 group, s(CH3), at 242 and 262 cm1 (marked by asterisks), and the deformation of the skeletal structure of Ala-Ala, d(skel.), at 401 cm1 (marked by square) are also observed. It is interesting to remember that a similar phenomenon was already observed in the L-valine crystal at low temperatures; the appearance of modes in the low-wavenumber region was associated with a phase transition undergone by the crystal between 100 and 120 K [23]. Such a phase transition observed in Ala-Ala can have an explanation similar to those presented for other amino acid crystals. The contraction of the unit cell due to cooling changes the intermolecular bonds of the crystal leading to modification of the crystal symmetry [23,24].
ðx yÞðz; zÞðx yÞ (cm1) – 92 – 114 154 243 262 304 319 339 376 391 402 468 553 651 679 731 802 852 887 912 955 1008 1049 1097 1118 1153 1260 1284 1320 1338 1369 1405 1451 1551 1602 1627 1681 2887 2904 2935 2974 2989 3012 3015 3216
Assignment ðx yÞðx þ y; zÞðx yÞ (cm1) 78 92 97 112 241 260 304 317 338 376 392 402 469 555 650 680 729 800 851 886 912 956 1009 1048 1097 1119 1151 1261 1283 1320 1338 1369 1405 1451 1551 1603 1627 1681 2884 2904 2933 2975 3013 3216
Lattice mode Lattice mode Lattice mode Lattice mode s(CO2 ) s(CH3) s(CH3) d(esq.) d(esq.) d(esq.) d(esq.) d(esq.) d(esq.) s(NH+3) r(CO 2) d(CO 2) do.p.(CONH) do.p.(CONH) x(CO2 ) r(CH3) + m(C–CH3) m(C–CH3) + m(C– NH+3) m(C–CO) + m(C–CO2 ) x(NH+3) x(NH+3) m(C–CH3) + d(CH3) x(CH3) m(CN) r(NH+3) d(CH) d(CH) d(CH) + d(NH) ds(CH3) ds(CH3) ms(CO2 ) da(CH3) d(NH) + m(CN) ma(CO2 ) da(NH+3) m(C@O) m(CH) ms(CH3) ms(CH3) ma(CH3) ma(CH3) ms(NH+3) ms(NH+3) ma(NH+3)
Additionally, it is worth to note that bands associated with CH3 group change as a consequence of the phase transition. In fact, an inelastic scattering study performed on L-alanine showed an anomaly of the mean-square displacement of the scattering nuclei between 160 and 220 K. Such an anomaly was interpreted as a phase transition (or a structural rearrangement) probably related to thermally activated reorientation of NH3 and CH3 groups [25]. In other words, while the CH3 group in L-alanine can be responsible for a structural rearrangement at low temperatures, it also seems to play some roles in Ala-Ala. This point will be discussed below. Another relevant point related to spectra in Fig. 3 concerns the band associated with rocking of CO 2 unit, r(CO2 ), observed at 555 cm1 at room temperature. Such a band in both scattering geometries studied in the present work can be fitted by one peak at high temperatures while at low temperatures two peaks are needed to fit it. This can be understood as a lower order for CO 2 groups at low temperatures.
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
(a)
247
(b)
Fig. 4. Raman spectra of Ala-Ala crystal in the 600–1200 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
(a)
(b)
Fig. 5. Raman spectra of Ala-Ala crystal in the 1200–1800 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
The Raman spectra of Ala-Ala crystal in both ðx yÞðz; zÞðx yÞ and ðx yÞðx þ y; zÞðx yÞ scattering geometries for several temperatures in the 600–1200 cm1 spectral region is shown in Fig. 4. In this region, one expects to observe vibrations associated + with bending of CO 2 unit, deformation of CONH, wagging of NH3 + and CO 2 and rocking of NH3 and CH3 groups, among others [26,27]. In this figure, modifications in the Raman spectrum similar to those noted in the previous spectral region are observed. As temperature decrease, one observes between 60 and 80 K the following phenomena: (i) the splitting of the band at 682 cm1 (marked by an arrow) which is associated with the out-of-plane deformation of CONH, do.p.(CONH); (ii) the splitting of the band marked by a star and associated with wagging vibration of CH3, x(CH3), located at 1096 cm1 at room temperature. It is worth also to mention the splitting of a band localized at 1050 cm1, which was tentatively associated with stretching of C–CH3 and bending of CH3, m(C–CH3) + d(CH3). In fact, between 300 and 100 K on cooling, the band shows a symmetric profile, but below 80 K it appears with an asymmetric shape; in the spectrum recorded at 11 K, the band splits into two peaks. These set of modifications can be seen
as additional evidences that Ala-Ala crystal undergoes a phase transition at T < 80 K. Fig. 5 shows the Raman spectra of Ala-Ala crystal in the spectral range 1200–1800 cm1 for several temperatures for two scattering geometries. In this spectral region, one expects to observe the bands associated with bending vibrations of NH, CH, CH3 and NH+3 units, as well as stretching vibrations of CO 2 and C@O units [23,28]. Bands at 1261 and 1283 cm1 are assigned as the deformation of CH, d(CH). The symmetric deformations of CH3 group, ds(CH3), are located at 1338 and 1369 cm1, while the anti-symmetric deformation of the same unit, da(CH3), is observed at 1451 cm1. The intense band observed at 1681 cm1 is associated with stretching of C@O, m(C@O), in accordance with several other studies on amino acid crystals [17,29,30]. From the analysis of temperature evolution of the bands in Fig. 5, it is also possible to observe the appearance of a band at 1301 cm1. The new band strongly supports the hypothesis of the occurrence of a phase transition undergone by Ala-Ala crystal. The clarity of the changes is in contrast with the behavior of the Raman spectra of other amino acid crystals in the same spectral region where the observed
248
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
(a)
(b)
Fig. 6. Raman spectra of Ala-Ala crystal in the 2700–3300 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
modifications are subtle: for example, although L-histidine presents a conformational phase transition at low temperatures, their Raman spectra between 1200 and 1800 cm1 do not show appearance of any new band nor splitting of peaks [28]. Fig. 6 presents the Raman spectra of Ala-Ala in the spectral region 2700–3300 cm1 for several temperatures for two scattering geometries. In this spectral region, it is possible to observe bands associated with stretching of CH, CH3 and NH+3 units (see the tentative assignment in Table 1) [17,26,27]. The changes in the Raman spectra upon variation of the temperature are not impressive. It is important to remember that, in general, great modifications in this spectral region could be associated with conformational changes of the molecules in the unit cell of the crystal [31,32]. In the case of the Ala-Ala crystal, only changes of intensities of the bands are observed. Such behavior is similar to that verified for DL-alanine crystal where only small change of intensities are observed at low temperatures [27]. However, differently from Ala-Ala crystal, in DL-alanine the modifications presented by the material is associated with slight changes of the CO 2 group orientation, not to a solid-solid phase transition. According to the group theory analysis, at room temperature, one expects to observe in the Raman spectrum 69 normal modes on each of the ðx yÞðz; zÞðx yÞ and ðx yÞðx þ y; zÞðx yÞ scattering geometries. Such a number is in agreement with the number of modes observed at room temperature and listed in Table 1. However, below 80 K, the number of modes observed in the Raman spectrum of Ala-Ala is increased. In the spectrum recorded at 20 K, one can clearly observe several new bands (at 132, 170, 176, 186, 195, 209, 216 and 222 cm1) in the lattice modes region. The total number of modes at low temperatures is not compatible with the C4 tetragonal structure. One possible way to explain the increase in the number of normal modes is supposing the occurrence of a phase transition from the tetragonal structure to a structure with lower symmetry. In particular, a monoclinic C2 symmetry is compatible with the number of normal modes observed in the Raman spectrum of Ala-Ala at low temperatures. Supposing a C4 ? C2 phase transition it is expected that the number of modes in the monoclinic phase be twice the number of modes in the tetragonal phase. When the group theory analysis for Ala-Ala is performed using the factor group C2, one obtains that the 276 degrees of freedom are now distributed as C = 138A + 138B. For the monocrystalline sample, it was possible to identify about 93 distinct modes (45 modes with A symmetry and 48
modes with E symmetry) in the spectrum of a polarized experiment. It is seen that the increasing of the number of observed modes is completely explained by group theory if we suppose the new symmetry C2 for the Ala-Ala at low temperatures. It is interesting to note that the changes were observed in almost all ranges of the Raman spectrum. By the analysis of Table 1, one sees that many of the important variations found in the spectra are related to the CH3 groups. In the presented work, it is suggested that the phase transition mechanism for this peptide crystal is the occupation of non-equivalents sites by the CH3 groups located in the center of the unit cell and those located near the borders, as seen in Fig. 2(b). At this point, it is worth to compare the phase transition undergone by Ala-Ala with the behavior of some simple amino acid crystals. For example, studies on the L-alanine crystal under low temperature do not present strong evidence that the material undergoes phase transition, although the splitting of infrared active bands at those conditions suggests that in the crystalline material there is a disorder due to protons occupying different positions in the hydrogen bonds formed by NH+3 group [33]. It is also interesting to remember that anomalies at low temperature were interpreted as opposite competing interactions from crystal packing forces and charge-phonon coupling like a Jahn–Teller effect [34]. The most important aspect is that although L-alanine exhibits some anomalies at low temperatures we can ascribe the orthorhombic room-temperature phase of L-alanine is stable down to 10 K. Similarly, when a-glycine is investigated under the scrutiny of Raman spectroscopy, no evidence of phase transition is observed under low-temperature conditions, although an increase in the interlayer hydrogen bond length is verified through the analysis of the N–H stretching vibration [35]. Another example of an amino acid crystal where it is observed a stable structure at low temperature is DL-serine. On the other hand, when L-serine was investigated by Raman spectroscopy, it was verified that the crystal undergoes a dynamic transition at 140 K [36]. This transition was interpreted as the orientation of the side chain–CH2OH fragments of the zwitterion with respect to the skeletal C–C bonds; as a result positional disorder of the O–H O intermolecular hydrogen bond was observed [36]. On the other hand, a Raman spectroscopic study showed that the amino acid L-valine seems to undergo a phase transition at 100 K, as indicated by the disappearance of a lattice mode for low temperatures [23]. This glance at the stability of some simple
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
amino acids at low temperatures highlights our result that in the simple dipeptide Ala-Ala, an instability is verified. Our Raman spectroscopic study indicates that the Ala-Ala crystal undergoes a continuous second order phase transition, going presumably from the tetragonal C4 structure to a monoclinic C2 structure. In such a phase transition one would expect the correlations (i) 69A + 69B from the C4 factor group to 138A in the C2 factor group and (ii) 69E from C4 symmetry to 138B in the C2 symmetry. Obviously, this is not definitive, future studies are needed to elucidate this issue conclusively. Conclusions In this work, the vibrational behavior of crystalline samples of Lalanyl-L-alanine was investigated as a function of temperature through Raman spectroscopy. A table with the tentative assignments for the Raman normal modes of vibration observed in the spectra for two different scattering geometries is presented. It was found that the sample remains stable down to a temperature of about 80 K, when it begins to undergo a slow second order phase transition from the tetragonal structure with factor group C4 to presumably a monoclinic structure with factor group C2. It is suggested that the governing mechanism of this transition is the occupation of non-equivalent sites by the CH3 groups present in the Ala-Ala molecule. Acknowledgments The authors would like to thank FUNCAP and CNPq Brazilian agencies for the financial support. References [1] T. Kimmerlin, D. Seebach, J. Pept. Res. 65 (2005) 229–260. [2] V.V. Suresh Babu, Resonance 16 (2011) 640–647. [3] E. Fischer, E. Fourneau, Berichte Der Dtsch. Chem. Gesellschaft 34 (1901) 2868–2877. [4] V. du Vigneaud, C. Ressler, J.M. Swan, C.W. Roberts, P.G. Katsoyannis, J. Am. Chem. Soc. 76 (1954) 3115–3121.
249
[5] A.J. Weaver, M.D. Kemple, F.G. Prendergast, Biophys. J. 54 (1988) 1–15. [6] A.I.R. Brewster, V.J. Hruby, A.F. Spatola, F.A. Bovey, Biochemistry 12 (1973) 1643–1649. [7] R. Lang, A.L. Gundlach, B. Kofler, Pharmacol. Ther. 115 (2007) 177–207. [8] B.E. Dunning, J.E. Foley, B. Ahrén, Diabetologia 48 (2005) 1700–1713. [9] V. Muñoz, L. Serrano, Nat. Struct. Biol. 1 (1994) 399–409. [10] J.T. López Navarrete, V. Hernández, F.J. Ramírez, J. Mol. Struct. 348 (1995) 249– 252. [11] R. Schweitzer-Stenner, F. Eker, Q. Huang, K. Griebenow, P.A. Mroz, P.M. Kozlowski, J. Phys. Chem. B 106 (2002) 4294–4304. [12] W.H. Moore, S. Krimm, Biopolymers 15 (1976) 2465–2483. [13] J.F. Rabolt, W.H. Moore, S. Krimm, Macromolecules 10 (1977) 1065–1074. [14] I. Compagnon, J. Oomens, J. Bakker, G. Meijer, G. von Helden, Phys. Chem. Chem. Phys. 7 (2005) 13–15. [15] Y.S. Kim, J. Wang, R.M. Hochstrasser, J. Phys. Chem. B. 109 (2005) 7511–7521. [16] J. Sebek, J. Kapitán, J. Sebestík, V. Baumruk, P. Bour, J. Phys. Chem. A 113 (2009) 7760–7768. [17] P. Bourˇ, J. Kapitán, V. Baumruk, J. Phys. Chem. A 105 (2001) 6362–6368. [18] T.C. Cheam, J. Mol. Struct. 295 (1993) 259–271. [19] R.J. Fletterick, C.C. Tsai, R.E. Hughes, J. Phys. Chem. 75 (1971) 918–922. [20] K. Momma, F. Izumi, J. Appl. Crystallogr. 44 (2011) 1272–1276. [21] D.L. Rousseau, R.P. Bauman, S.P.S. Porto, J. Raman Spectrosc. 10 (1981) 253– 290. [22] B.T.O. Abagaro, P.T.C. Freire, J.G. Silva, F.E.A. Melo, J.A. Lima Jr., J. Mendes Filho, P.S. Pizani, Vib. Spectro. 66 (2013) 119–122. [23] J.A. Lima Jr., P.T.C. Freire, R.J.C. Lima, A.J.D. Moreno, J. Mendes Filho, F.E.A. Melo, J. Raman Spectrosc. 36 (2005) 1076–1081. [24] S.V. Goryainov, E.N. Kolesnik, E.V. Boldyreva, Phys. B Condens. Matter. 357 (2005) 340–347. [25] J.M. de Souza, P.T.C. Freire, H.N. Bordallo, D.N. Argyriou, J. Phys. Chem. B 111 (2007) 5034–5039. [26] G.P. De Sousa, P.T.C. Freire, J.A. Lima, J.M. Filho, F.E.A. Melo, Vib. Spectrosc. 57 (2011) 102–107. [27] J.A. Lima Jr., P.T.C. Freire, F.E.A. Melo, J. Mendes Filho, G.P. De Sousa, R.J.C. Lima, P.F. Façanha Filho, H.N. Bordallo, J. Raman Spectrosc. 41 (2010) 808–813. [28] G.P. De Sousa, P.T.C. Freire, J.M. Filho, F.E.A. Melo, C.L. Lima, Brazilian, J. Phys. 43 (2013) 137–144. [29] S. Celik, A.E. Ozel, S. Kecel, S. Akyuz, Vib. Spectrosc. 61 (2012) 54–65. [30] M. Wolpert, P. Hellwig, Spectrochim. Acta A 64 (2006) 987–1001. [31] X. Cao, G. Fischer, J. Phys. Chem. A 106 (2002) 41–50. [32] J.A. Lima Jr., P.T.C. Freire, F.E.A. Melo, V. Lemos, J. Mendes Filho, P.S. Pizani, J. Raman Spectrosc. 39 (2008) 1356–1363. [33] M. Rozenberg, G. Shoham, I. Reva, R. Fausto, Spectrochim. Acta A 59 (2003) 3253–3266. [34] M. Barthes, H.N. Bordallo, F. Dénoyer, J.-E. Lorenzo, J. Zaccaro, A. Robert, F. Zontone, Eur. Phys. J. B – Condens. Matter. 37 (2003) 375–382. [35] C. Murli, S. Thomas, S. Venkateswaran, S.M. Sharma, Phys. B Condens. Matter. 364 (2005) 233–238. [36] B.A. Kolesov, E.V. Boldyreva, J. Phys. Chem. B 111 (2007) 14387–14397.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
The temperature-dependent single-crystal Raman spectroscopy of a model dipeptide: L-Alanyl-L-alanine J.G. Silva a, L.M. Arruda a, G.S. Pinheiro a,b,⇑, C.L. Lima b, F.E.A. Melo a, A.P. Ayala a, J. Mendes Filho a, P.T.C. Freire a a b
Departamento de Física, Universidade Federal do Ceará, C.P. 6030, CEP 60455-760 Fortaleza, CE, Brazil Departamento de Física, Campus Ministro Petrônio Portella, Universidade Federal do Piauí, CEP 64049-550 Teresina, PI, Brazil
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
Temperature dependent polarized
Raman spectra of the L-alanyl-Lalanine. The crystal of L-alanyl-L-alanine undergoes a structural phase transition. The phase transition is observed between 80 and 60 K.
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 4 April 2015 Accepted 7 April 2015 Available online 11 April 2015 Keywords: Raman spectroscopy Infrared spectroscopy Peptide Phase transition
a b s t r a c t A single-crystal of peptide L-alanyl-L-alanine (C6H12N2O3) was studied by Raman spectroscopy at lowtemperature, and a tentative assignment of the normal modes was given. Evidence of a second order structural phase transition was found through Raman spectroscopy between the temperatures of 80 K and 60 K. Group theory considerations suggest that the transition leads the sample from the tetragonal to a monoclinic structure. Additionally, our study suggests that the mechanism for the structural phase transition is governed by the occupation of non-equivalent C1 local symmetry sites by the CH3 molecular groups. Analysis based on group theory suggests L-alanyl-L-alanine presents C2 symmetry at low temperatures. Ó 2015 Elsevier B.V. All rights reserved.
Introduction Peptides are important organic compounds not only because of their ability to form proteins but because of the central role they play in several biological and physiological processes in living organisms [1]. Among the biological functions displayed by peptides are their capabilities as neurotransmitters and hormones. ⇑ Corresponding author at: Departamento de Física, Universidade Federal do Ceará, C.P. 6030, CEP 60455-760 Fortaleza, CE, Brazil. Tel.: +55 8533669906; fax: +55 8533669450. E-mail address: gardenia@fisica.ufc.br (G.S. Pinheiro). http://dx.doi.org/10.1016/j.saa.2015.04.010 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Additionally, they are important for being constituents of several protein chains and taking part in our biological metabolism. As a consequence, peptides have received special attention from researchers of the pharmacological area. Further uses as, for instance, antibiotic and immunological agents are also active fields of study for peptides [2]. As a consequence, the study of peptides has considerably grown in importance in the last few decades. Although its origin is credited to Emil Fischer for his article on the synthesis of the dipeptide glycylglycine in 1901 [3], the subject remained relatively dormant for years due to difficulties the peptide synthesis presented. This difficulty was solved gradually with increasingly efficient
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
245
found in the literature, although some works performed at room temperature were presented [15–18]. The understanding of vibrational and structural properties of peptides is of relevance since they have potential use in medicine. In this work, the vibrational properties of Ala-Ala through Raman spectroscopy at low temperatures are investigated, and an assignment of the vibrational modes is provided. Careful analysis of the behavior of the vibrational modes as a function of temperature is also furnished. Fig. 1. Experimental and calculated powder X-ray diffractogram of L-alanyl-Lalanine crystal.
Experimental procedures
temporary protections for the amino group. After the synthesis of an active hormone, the octapeptide oxytocin, by du Vigneaud et al. in 1954 [4], the variety of synthesized natural occurring peptides and analogs grew along with the demand for them and the study of their properties. The study of peptides is varied in scope and methods [5–9]. Vibrational spectroscopy has become an important tool for the investigation of protein and peptide science and as such, several studies on vibrational properties of peptides have been conducted [10–14]. L-Alanyl-L-alanine (Ala-Ala) is one of the simplest peptides but any study of its vibrational properties at low temperatures are
Single-crystal samples of Ala-Ala (C6H12N2O3) were grown by the vapor-diffusion technique at controlled temperature (278 K). The crystals obtained were thin platelets shape and colorless. The crystal structure of the samples obtained was confirmed through an X-ray powder diffraction experiment using a D8 Advance (Bruker) diffractometer using Cu Ka radiation operating at 40 kV, 40 mA. The Raman spectra of Ala-Ala were obtained using a T64000 Jobin Yvon spectrometer equipped with a liquid N2-cooled charged coupled device detection system and a 514.5 nm argon ion laser. The slit of the spectrometer allowed a resolution of 2 cm1. In order to obtain low temperatures, the sample was placed in a helium flux cryostat (model DE202S, from Air Products and Chemicals), and the temperature was monitored in the interval
Fig. 2. (a) Molecular structure of Ala-Ala; (b) unit cell of Ala-Ala crystal.
(a)
(b)
Fig. 3. Raman spectra of Ala-Ala crystal in the 50–600 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
246
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
20–300 K by a digital temperature controller unit having a stability of 0.1 K.
Scattering geometry
Results and discussion Ala-Ala crystallizes in the tetragonal structure belonging to the I4 (C54) space group [19]. The lattice parameters are a = b = 17.985 Å and c = 5.154 Å, and contains eight formulas per unit cell. Fig. 1 shows the Ala-Ala X-ray diffraction pattern obtained at room temperature, refined by Rietveld method with R-factors Rwp = 10.3% and S = 1.2 for the goodness of fit. The data obtained is good agreement with the pattern previously published [19]. The molecular structure and a view of the unit cell are shown in Fig. 2(a) and (b). The structures were visualized by VESTA [20]. The molecular packaging of Ala-Ala is formed by eight hydrogen bonds between the nitrogen of one molecule with carboxylate oxygens from four different molecules. Factor group analysis shows that the 273 optical modes predicted decompose into the irreducible representations as C = 68A + 69B + 68E. All representations are Raman active and the A and E representations are also infrared active. The axes of the crystal were defined according to the following convention: the z-axis was that perpendicular to the plane of the platelet, the y-axis was defined as the axis coincident with the longest dimension of the crystal and the x-axis was defined perpendicular to the y- and z-axes. The Raman spectra were taken for
two
scattering
Table 1 Vibrational wavenumbers (cm1) and tentative assignment for the Ala-Ala from spectra recorded at room temperature.
geometries:
ðx yÞðz; zÞðx yÞ
and
ðx yÞðx þ y; zÞðx yÞ, which provide data on the modes with A and E symmetries, respectively. In this work, the usual Porto’s notation [21] was used for describing the scattering geometries. Because of the platelet form of the crystals obtained, the z(xy)z geometry was not available, and the phonons of the B irreducible representation are not presented in this work. Fig. 3 shows the Raman spectra of Ala-Ala for several temperatures in the spectral range 50–600 cm1, for (a) the ðx yÞðz; zÞðx yÞ and (b) the ðx yÞðx þ y; zÞðx yÞ scattering geometries. All the spectra presented in this work were taken in the cooling cycle. This region is characterized by the external modes of the crystal and by internal modes of low-energy. These bands provide important insights about the structural stability of the material [22–24]. The band at 153 cm1 is associated with 1 the torsion of CO is 2 unit, s(CO2 ), and the band at 242 cm assigned as the torsion of CH3, s(CH3). A band of great importance due to its relationship with the hydrogen bond is torsion of NH+3, s(NH+3), which is observed at 473 cm1 in the Raman spectrum. Table 1 presents the tentative assignment of the bands observed in the Raman spectra of Ala-Ala at room temperature for the two scattering geometries. Under cooling, the Raman spectra in Fig. 3 show great changes in both scattering geometries between 60 and 80 K. An increase in the number of the modes in the region 50–200 cm1 is observed indicating a change of symmetry of the crystal. Furthermore, in the region 200–600 cm1, the splitting of bands associated with both the torsion of CH3 group, s(CH3), at 242 and 262 cm1 (marked by asterisks), and the deformation of the skeletal structure of Ala-Ala, d(skel.), at 401 cm1 (marked by square) are also observed. It is interesting to remember that a similar phenomenon was already observed in the L-valine crystal at low temperatures; the appearance of modes in the low-wavenumber region was associated with a phase transition undergone by the crystal between 100 and 120 K [23]. Such a phase transition observed in Ala-Ala can have an explanation similar to those presented for other amino acid crystals. The contraction of the unit cell due to cooling changes the intermolecular bonds of the crystal leading to modification of the crystal symmetry [23,24].
ðx yÞðz; zÞðx yÞ (cm1) – 92 – 114 154 243 262 304 319 339 376 391 402 468 553 651 679 731 802 852 887 912 955 1008 1049 1097 1118 1153 1260 1284 1320 1338 1369 1405 1451 1551 1602 1627 1681 2887 2904 2935 2974 2989 3012 3015 3216
Assignment ðx yÞðx þ y; zÞðx yÞ (cm1) 78 92 97 112 241 260 304 317 338 376 392 402 469 555 650 680 729 800 851 886 912 956 1009 1048 1097 1119 1151 1261 1283 1320 1338 1369 1405 1451 1551 1603 1627 1681 2884 2904 2933 2975 3013 3216
Lattice mode Lattice mode Lattice mode Lattice mode s(CO2 ) s(CH3) s(CH3) d(esq.) d(esq.) d(esq.) d(esq.) d(esq.) d(esq.) s(NH+3) r(CO 2) d(CO 2) do.p.(CONH) do.p.(CONH) x(CO2 ) r(CH3) + m(C–CH3) m(C–CH3) + m(C– NH+3) m(C–CO) + m(C–CO2 ) x(NH+3) x(NH+3) m(C–CH3) + d(CH3) x(CH3) m(CN) r(NH+3) d(CH) d(CH) d(CH) + d(NH) ds(CH3) ds(CH3) ms(CO2 ) da(CH3) d(NH) + m(CN) ma(CO2 ) da(NH+3) m(C@O) m(CH) ms(CH3) ms(CH3) ma(CH3) ma(CH3) ms(NH+3) ms(NH+3) ma(NH+3)
Additionally, it is worth to note that bands associated with CH3 group change as a consequence of the phase transition. In fact, an inelastic scattering study performed on L-alanine showed an anomaly of the mean-square displacement of the scattering nuclei between 160 and 220 K. Such an anomaly was interpreted as a phase transition (or a structural rearrangement) probably related to thermally activated reorientation of NH3 and CH3 groups [25]. In other words, while the CH3 group in L-alanine can be responsible for a structural rearrangement at low temperatures, it also seems to play some roles in Ala-Ala. This point will be discussed below. Another relevant point related to spectra in Fig. 3 concerns the band associated with rocking of CO 2 unit, r(CO2 ), observed at 555 cm1 at room temperature. Such a band in both scattering geometries studied in the present work can be fitted by one peak at high temperatures while at low temperatures two peaks are needed to fit it. This can be understood as a lower order for CO 2 groups at low temperatures.
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
(a)
247
(b)
Fig. 4. Raman spectra of Ala-Ala crystal in the 600–1200 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
(a)
(b)
Fig. 5. Raman spectra of Ala-Ala crystal in the 1200–1800 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
The Raman spectra of Ala-Ala crystal in both ðx yÞðz; zÞðx yÞ and ðx yÞðx þ y; zÞðx yÞ scattering geometries for several temperatures in the 600–1200 cm1 spectral region is shown in Fig. 4. In this region, one expects to observe vibrations associated + with bending of CO 2 unit, deformation of CONH, wagging of NH3 + and CO 2 and rocking of NH3 and CH3 groups, among others [26,27]. In this figure, modifications in the Raman spectrum similar to those noted in the previous spectral region are observed. As temperature decrease, one observes between 60 and 80 K the following phenomena: (i) the splitting of the band at 682 cm1 (marked by an arrow) which is associated with the out-of-plane deformation of CONH, do.p.(CONH); (ii) the splitting of the band marked by a star and associated with wagging vibration of CH3, x(CH3), located at 1096 cm1 at room temperature. It is worth also to mention the splitting of a band localized at 1050 cm1, which was tentatively associated with stretching of C–CH3 and bending of CH3, m(C–CH3) + d(CH3). In fact, between 300 and 100 K on cooling, the band shows a symmetric profile, but below 80 K it appears with an asymmetric shape; in the spectrum recorded at 11 K, the band splits into two peaks. These set of modifications can be seen
as additional evidences that Ala-Ala crystal undergoes a phase transition at T < 80 K. Fig. 5 shows the Raman spectra of Ala-Ala crystal in the spectral range 1200–1800 cm1 for several temperatures for two scattering geometries. In this spectral region, one expects to observe the bands associated with bending vibrations of NH, CH, CH3 and NH+3 units, as well as stretching vibrations of CO 2 and C@O units [23,28]. Bands at 1261 and 1283 cm1 are assigned as the deformation of CH, d(CH). The symmetric deformations of CH3 group, ds(CH3), are located at 1338 and 1369 cm1, while the anti-symmetric deformation of the same unit, da(CH3), is observed at 1451 cm1. The intense band observed at 1681 cm1 is associated with stretching of C@O, m(C@O), in accordance with several other studies on amino acid crystals [17,29,30]. From the analysis of temperature evolution of the bands in Fig. 5, it is also possible to observe the appearance of a band at 1301 cm1. The new band strongly supports the hypothesis of the occurrence of a phase transition undergone by Ala-Ala crystal. The clarity of the changes is in contrast with the behavior of the Raman spectra of other amino acid crystals in the same spectral region where the observed
248
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
(a)
(b)
Fig. 6. Raman spectra of Ala-Ala crystal in the 2700–3300 cm1 spectral region for several temperatures for the (a) ðx yÞðz; zÞðx yÞ and (b) ðx yÞðx þ y; zÞðx yÞ scattering geometries.
modifications are subtle: for example, although L-histidine presents a conformational phase transition at low temperatures, their Raman spectra between 1200 and 1800 cm1 do not show appearance of any new band nor splitting of peaks [28]. Fig. 6 presents the Raman spectra of Ala-Ala in the spectral region 2700–3300 cm1 for several temperatures for two scattering geometries. In this spectral region, it is possible to observe bands associated with stretching of CH, CH3 and NH+3 units (see the tentative assignment in Table 1) [17,26,27]. The changes in the Raman spectra upon variation of the temperature are not impressive. It is important to remember that, in general, great modifications in this spectral region could be associated with conformational changes of the molecules in the unit cell of the crystal [31,32]. In the case of the Ala-Ala crystal, only changes of intensities of the bands are observed. Such behavior is similar to that verified for DL-alanine crystal where only small change of intensities are observed at low temperatures [27]. However, differently from Ala-Ala crystal, in DL-alanine the modifications presented by the material is associated with slight changes of the CO 2 group orientation, not to a solid-solid phase transition. According to the group theory analysis, at room temperature, one expects to observe in the Raman spectrum 69 normal modes on each of the ðx yÞðz; zÞðx yÞ and ðx yÞðx þ y; zÞðx yÞ scattering geometries. Such a number is in agreement with the number of modes observed at room temperature and listed in Table 1. However, below 80 K, the number of modes observed in the Raman spectrum of Ala-Ala is increased. In the spectrum recorded at 20 K, one can clearly observe several new bands (at 132, 170, 176, 186, 195, 209, 216 and 222 cm1) in the lattice modes region. The total number of modes at low temperatures is not compatible with the C4 tetragonal structure. One possible way to explain the increase in the number of normal modes is supposing the occurrence of a phase transition from the tetragonal structure to a structure with lower symmetry. In particular, a monoclinic C2 symmetry is compatible with the number of normal modes observed in the Raman spectrum of Ala-Ala at low temperatures. Supposing a C4 ? C2 phase transition it is expected that the number of modes in the monoclinic phase be twice the number of modes in the tetragonal phase. When the group theory analysis for Ala-Ala is performed using the factor group C2, one obtains that the 276 degrees of freedom are now distributed as C = 138A + 138B. For the monocrystalline sample, it was possible to identify about 93 distinct modes (45 modes with A symmetry and 48
modes with E symmetry) in the spectrum of a polarized experiment. It is seen that the increasing of the number of observed modes is completely explained by group theory if we suppose the new symmetry C2 for the Ala-Ala at low temperatures. It is interesting to note that the changes were observed in almost all ranges of the Raman spectrum. By the analysis of Table 1, one sees that many of the important variations found in the spectra are related to the CH3 groups. In the presented work, it is suggested that the phase transition mechanism for this peptide crystal is the occupation of non-equivalents sites by the CH3 groups located in the center of the unit cell and those located near the borders, as seen in Fig. 2(b). At this point, it is worth to compare the phase transition undergone by Ala-Ala with the behavior of some simple amino acid crystals. For example, studies on the L-alanine crystal under low temperature do not present strong evidence that the material undergoes phase transition, although the splitting of infrared active bands at those conditions suggests that in the crystalline material there is a disorder due to protons occupying different positions in the hydrogen bonds formed by NH+3 group [33]. It is also interesting to remember that anomalies at low temperature were interpreted as opposite competing interactions from crystal packing forces and charge-phonon coupling like a Jahn–Teller effect [34]. The most important aspect is that although L-alanine exhibits some anomalies at low temperatures we can ascribe the orthorhombic room-temperature phase of L-alanine is stable down to 10 K. Similarly, when a-glycine is investigated under the scrutiny of Raman spectroscopy, no evidence of phase transition is observed under low-temperature conditions, although an increase in the interlayer hydrogen bond length is verified through the analysis of the N–H stretching vibration [35]. Another example of an amino acid crystal where it is observed a stable structure at low temperature is DL-serine. On the other hand, when L-serine was investigated by Raman spectroscopy, it was verified that the crystal undergoes a dynamic transition at 140 K [36]. This transition was interpreted as the orientation of the side chain–CH2OH fragments of the zwitterion with respect to the skeletal C–C bonds; as a result positional disorder of the O–H O intermolecular hydrogen bond was observed [36]. On the other hand, a Raman spectroscopic study showed that the amino acid L-valine seems to undergo a phase transition at 100 K, as indicated by the disappearance of a lattice mode for low temperatures [23]. This glance at the stability of some simple
J.G. Silva et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 244–249
amino acids at low temperatures highlights our result that in the simple dipeptide Ala-Ala, an instability is verified. Our Raman spectroscopic study indicates that the Ala-Ala crystal undergoes a continuous second order phase transition, going presumably from the tetragonal C4 structure to a monoclinic C2 structure. In such a phase transition one would expect the correlations (i) 69A + 69B from the C4 factor group to 138A in the C2 factor group and (ii) 69E from C4 symmetry to 138B in the C2 symmetry. Obviously, this is not definitive, future studies are needed to elucidate this issue conclusively. Conclusions In this work, the vibrational behavior of crystalline samples of Lalanyl-L-alanine was investigated as a function of temperature through Raman spectroscopy. A table with the tentative assignments for the Raman normal modes of vibration observed in the spectra for two different scattering geometries is presented. It was found that the sample remains stable down to a temperature of about 80 K, when it begins to undergo a slow second order phase transition from the tetragonal structure with factor group C4 to presumably a monoclinic structure with factor group C2. It is suggested that the governing mechanism of this transition is the occupation of non-equivalent sites by the CH3 groups present in the Ala-Ala molecule. Acknowledgments The authors would like to thank FUNCAP and CNPq Brazilian agencies for the financial support. References [1] T. Kimmerlin, D. Seebach, J. Pept. Res. 65 (2005) 229–260. [2] V.V. Suresh Babu, Resonance 16 (2011) 640–647. [3] E. Fischer, E. Fourneau, Berichte Der Dtsch. Chem. Gesellschaft 34 (1901) 2868–2877. [4] V. du Vigneaud, C. Ressler, J.M. Swan, C.W. Roberts, P.G. Katsoyannis, J. Am. Chem. Soc. 76 (1954) 3115–3121.
249
[5] A.J. Weaver, M.D. Kemple, F.G. Prendergast, Biophys. J. 54 (1988) 1–15. [6] A.I.R. Brewster, V.J. Hruby, A.F. Spatola, F.A. Bovey, Biochemistry 12 (1973) 1643–1649. [7] R. Lang, A.L. Gundlach, B. Kofler, Pharmacol. Ther. 115 (2007) 177–207. [8] B.E. Dunning, J.E. Foley, B. Ahrén, Diabetologia 48 (2005) 1700–1713. [9] V. Muñoz, L. Serrano, Nat. Struct. Biol. 1 (1994) 399–409. [10] J.T. López Navarrete, V. Hernández, F.J. Ramírez, J. Mol. Struct. 348 (1995) 249– 252. [11] R. Schweitzer-Stenner, F. Eker, Q. Huang, K. Griebenow, P.A. Mroz, P.M. Kozlowski, J. Phys. Chem. B 106 (2002) 4294–4304. [12] W.H. Moore, S. Krimm, Biopolymers 15 (1976) 2465–2483. [13] J.F. Rabolt, W.H. Moore, S. Krimm, Macromolecules 10 (1977) 1065–1074. [14] I. Compagnon, J. Oomens, J. Bakker, G. Meijer, G. von Helden, Phys. Chem. Chem. Phys. 7 (2005) 13–15. [15] Y.S. Kim, J. Wang, R.M. Hochstrasser, J. Phys. Chem. B. 109 (2005) 7511–7521. [16] J. Sebek, J. Kapitán, J. Sebestík, V. Baumruk, P. Bour, J. Phys. Chem. A 113 (2009) 7760–7768. [17] P. Bourˇ, J. Kapitán, V. Baumruk, J. Phys. Chem. A 105 (2001) 6362–6368. [18] T.C. Cheam, J. Mol. Struct. 295 (1993) 259–271. [19] R.J. Fletterick, C.C. Tsai, R.E. Hughes, J. Phys. Chem. 75 (1971) 918–922. [20] K. Momma, F. Izumi, J. Appl. Crystallogr. 44 (2011) 1272–1276. [21] D.L. Rousseau, R.P. Bauman, S.P.S. Porto, J. Raman Spectrosc. 10 (1981) 253– 290. [22] B.T.O. Abagaro, P.T.C. Freire, J.G. Silva, F.E.A. Melo, J.A. Lima Jr., J. Mendes Filho, P.S. Pizani, Vib. Spectro. 66 (2013) 119–122. [23] J.A. Lima Jr., P.T.C. Freire, R.J.C. Lima, A.J.D. Moreno, J. Mendes Filho, F.E.A. Melo, J. Raman Spectrosc. 36 (2005) 1076–1081. [24] S.V. Goryainov, E.N. Kolesnik, E.V. Boldyreva, Phys. B Condens. Matter. 357 (2005) 340–347. [25] J.M. de Souza, P.T.C. Freire, H.N. Bordallo, D.N. Argyriou, J. Phys. Chem. B 111 (2007) 5034–5039. [26] G.P. De Sousa, P.T.C. Freire, J.A. Lima, J.M. Filho, F.E.A. Melo, Vib. Spectrosc. 57 (2011) 102–107. [27] J.A. Lima Jr., P.T.C. Freire, F.E.A. Melo, J. Mendes Filho, G.P. De Sousa, R.J.C. Lima, P.F. Façanha Filho, H.N. Bordallo, J. Raman Spectrosc. 41 (2010) 808–813. [28] G.P. De Sousa, P.T.C. Freire, J.M. Filho, F.E.A. Melo, C.L. Lima, Brazilian, J. Phys. 43 (2013) 137–144. [29] S. Celik, A.E. Ozel, S. Kecel, S. Akyuz, Vib. Spectrosc. 61 (2012) 54–65. [30] M. Wolpert, P. Hellwig, Spectrochim. Acta A 64 (2006) 987–1001. [31] X. Cao, G. Fischer, J. Phys. Chem. A 106 (2002) 41–50. [32] J.A. Lima Jr., P.T.C. Freire, F.E.A. Melo, V. Lemos, J. Mendes Filho, P.S. Pizani, J. Raman Spectrosc. 39 (2008) 1356–1363. [33] M. Rozenberg, G. Shoham, I. Reva, R. Fausto, Spectrochim. Acta A 59 (2003) 3253–3266. [34] M. Barthes, H.N. Bordallo, F. Dénoyer, J.-E. Lorenzo, J. Zaccaro, A. Robert, F. Zontone, Eur. Phys. J. B – Condens. Matter. 37 (2003) 375–382. [35] C. Murli, S. Thomas, S. Venkateswaran, S.M. Sharma, Phys. B Condens. Matter. 364 (2005) 233–238. [36] B.A. Kolesov, E.V. Boldyreva, J. Phys. Chem. B 111 (2007) 14387–14397.