Abstract
In this chapter, we investigate the Raman spectra of proteinogenic amino acid crystals. Amino acids are fundamental organic molecules that compose polypeptides (a linear chain of amino acids) and proteins (folded polypeptides with specific functions) found in all living beings. Surprisingly, the number of these basic molecules is not more than 22 (20 of them commonly known as the standard amino acids, plus pyrrolysine and selenocysteine). They are defined as a molecule formed by an NH2 group, a COOH group, a lateral chain (the R group), and a hydrogen atom, all of them connected to a single carbon, the α-carbon. Interestingly, α-amino acids show chirality, i.e., they present different distributions of group of atoms around the α-carbon, being defined as l- and d-form. For amino acids and proteins found in the living beings, the l-form is the dominant form, although some exceptions have been discovered in the last decades. In this chapter, we present the Raman spectra of all standard amino acids and discuss the different kinds of vibrations found, comparing them. As complementary part of the work, we present results on vibrational properties of some amino acids using Raman spectroscopy when subjected to specific conditions, with variation in temperature or pressure. Finally, we present some perspectives as the investigation of purines, a group of molecules associated with the DNA molecule.
Keywords
- amino acid
- raman spectroscopy
- vibrational property
1. Introduction
Amino acids constitute an impressive and mysterious class of organic molecules, impressive because they form all proteins of living beings and mysterious because of their simplicity. In fact, amino acids — in their α form and in the
As it is well established, every cell of the living being on the Earth uses a set of 20 amino acids to produce all kinds of proteins [1, 2]. The 20 standard amino acids can be classified as (i) unpolar (alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan) and (ii) polar (glycine, arginine, asparagine, cysteine, glutamine, lysine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and histidine). Additionally, some compendia include selenocysteine and pyrrolysine as belonging to the group of proteinogenic amino acids, but this is not unanimity yet. In the last years, a series of studies have investigated the vibrational and structural properties of amino acid crystals [3–42].
One of the main techniques to investigate vibrational properties of materials, whatever it is, is Raman spectroscopy. The technique consists in the interaction of light from a laser source with the material and further scattering of the light. The scattered light carries information about the rotational, vibrational, and, eventually, electronic states of the material. Concerning the Raman scattering effect, a pivotal concept is the scattering cross-section, σ, which represents a likelihood of a scattering event to occur [43]. It is defined as the rate at which energy is removed from the incident photon by the scattering substance, divided by the rate at which energy in the incident photon crosses a unit area perpendicular to its direction of propagation [43], expressed as in Eq. (1):
In this equation,
with the sum performed over all possible eigenstates
In this expression, the kets |
In this chapter, we summarize the main studies on Raman spectroscopy applied to proteinogenic amino acid molecules and crystals and point new perspectives on the subject.
2. Experimental details
In the present work, we have used two experimental set-ups: an instrument using Fourier transform mechanism and a dispersive (conventional) spectrometer. On the one hand, FT-Raman spectra were recorded using a Bruker RFS100/S FTR system and a D418-T detector, with the sample excited by means of the 1064 nm line of a Nd:YAG laser. In these cases, the spectral resolution was 4 cm−1. On the other hand, the conventional Raman spectra were excited with the 514.5 nm line of argon ion lasers and the scattered light was analyzed in a Jobin-Yvon T64000 spectrometer equipped with the nitrogen cooled CCD system. Typically, the spectral resolution in the conventional Raman experiments was 2 cm−1. Theoretically, the bands appearing in the Raman spectrum are independent of the excitation energy of the laser, although the intensity of the scattered light is proportional to
3. Nonpolar amino acids
In the discussion, we will separate the discussion according to the polarity of the lateral groups of the amino acids. We begin with the apolar amino acids. This group is composed of the following amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and proline.
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig1.png)
Figure 1.
Polarized Raman spectra of glycine (α-form) for two scattering geometries in the high wavenumber region of the spectrum.
Although the chirality itself is a theme of great relevance, in the present chapter we discuss only the properties of the l-chiral sister of amino acid crystals (those present in the proteins) and do not furnish further information about the phenomenon. The simplest chiral amino acid is
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig2.png)
Figure 2.
Raman spectrum of
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig3.png)
Figure 3.
FT-Raman spectra of
Figure 3 shows the FT-Raman spectrum of
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig4.png)
Figure 4.
Raman spectra of
Another amino acid with nonpolar characteristics is
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig5.png)
Figure 5.
FT-Raman spectra of
Figure 5 presents the FT-Raman spectrum of
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig6.png)
Figure 6.
FT-Raman spectra of
The Raman spectra of
4. Neutral, polar amino acids
Among the polar amino acids, serine, cysteine, asparagine, glutamine, threonine, and tyrosine are neutral. Figure 7 shows the Raman spectra of l-cysteine.HCl, l-serine, l-glutamine, and l-asparagine.H2O as obtained through a Fourier-transform Raman spectrometer in the spectral range from 50 to 3500 cm−1. From 1800 to 2800 cm−1 no mode is observed, except in the spectrum of l-cysteine.HCl, where a stretching vibration of SH appears at ~ 2550 cm−1. In fact, as mentioned in the previous paragraph, cysteine is the only proteinogenic amino acid that presents an S-H bond and, as a consequence, is the only amino acid to present a peak in this region.
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig7.png)
Figure 7.
FT-Raman spectra of polar (neutral) amino acids
It is important to remember that
Obviously, if you change the sulfur atom by an oxygen atom, the hydrogen bonds involving SH groups cease to exist. As a molecule,
The radical NH2-(C=O)-CH2 characterizes the amino acid l-
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig8.png)
Figure 8.
FT-Raman spectrum of
The FT-Raman spectrum of
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig9.png)
Figure 9.
FT-Raman spectrum of
Figure 9 shows the FT-Raman spectrum of
5. Acidic, polar amino acids
Figure 10 shows the FT-Raman spectra of
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig10.png)
Figure 10.
FT-Raman spectra of
6. Basic, polar amino acids
l-histidine was investigated through Raman spectroscopy in a recent paper that explored the vibrational behavior of the crystal under cryogenic conditions [41]. l-histidine can crystallize in two different polymorphs with monoclinic or orthorhombic symmetry. The work of reference [41] has investigated the orthorhombic form of the crystal that presents a
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig11.png)
Figure 11.
Raman spectra of
Up to now, the Raman scattering investigations have furnished an interesting picture about the vibrational aspects of diverse amino acids. Some studies have even studied the behavior of the crystals under extreme conditions, low temperature or high pressure. However, a complete understanding involving connections, for example, between the hydrogen bonds and the physical properties of the crystal is still lacking. Obviously, some preliminary attempts are already known, such as a possible connection between the dimensions of hydrogen bonds and the behavior of torsional vibration of NH3+ under high pressure (for l-alanine, l-threonine, and taurine [17]). A fundamental question in biochemistry is to realize why the proteins of all living beings are formed by the l-form of amino acids (the d-form is found only isolated in the plasma of certain cells). Some glimpses were given by Abdus Salam who speculates the occurrence of a phase transition explained through BCS theory, gauge field theory, and Higgs mechanism [44]. There is also suggestion that ultraviolet radiation should be able to select one of the chiral forms of the amino acid, but, in fact, all these suggestions are suppositions waiting for confirmation. This problem deserves future investigations. But, is the behavior of d-amino acid crystals the same of l-amino acids under extreme conditions? At first, the answer to this question should be positive because both l- and d-forms of the amino acids are equivalent from an energetic point of view. However, some preliminary results point to different behavior for the two forms in some special cases, but we do not have space to discuss such intriguing point in this chapter. Maybe, surprising information is waiting for us in the coming years.
7. Beyond amino acids
The success obtained by the investigation of amino acids has incentivized the study of other simple organic molecules of living beings. After furnishing a more or less closed picture about amino acids, the next natural step is the study of peptides, but we will not discuss them in this chapter. We prefer to analyze another natural choose, molecules involved in the DNA structure. One example we will explore in this chapter is thymidine, a nucleoside constituted of a deoxyribose and the pyrimidine base thymine. It is found in the DNA of all living organisms. The Raman spectrum presents a very intense set of bands in the low wavenumber region that are associated with the lattice modes (Figure 12). This is very interesting because in future analysis of the crystal under extreme conditions, the behavior of the lattice modes should be a pivotal point in order to understand eventual structural modification. A strong band observed at 1665 cm−1 is assigned as in-plane vibration involving C = O and C = C and a band at 1690 cm−1 is assigned as stretching C = O, ν(C=O). Bending of CH3, δ(CH3), is identified as the band at 1438, 1457, and 1480 cm−1. The band observed at 1031 cm−1 is associated with bending of CNH, δ(CNH), and the band at 1000 cm−1 is associated with bending OCH, δ(OCH). An out-of-plane vibration involving CH is observed at 972 cm−1 and a pyrimidine ring breathing is observed at 773 cm−1. Additionally, out-of-plane vibration involving CCH3 group is observed at 396 and 378 cm−1 and in plane vibration involving the same group is observed at 276 and 306 cm−1. In the high wavenumber region of the Raman spectrum is possible to observe a series of bands, among them one observed at 3298 cm−1 that was assigned as stretching of OH, ν(OH). A series of bands is observed at 2952, 2965, 2973, and 2991 cm−1 and they are classified as stretching of CH, CH2, and CH3 units. Finally, let us single out an important point related to the study of thymidine, its behavior as a function of temperature. In order to make the presentation of this section more complete, we have performed study of thymidine crystal under low temperature. Analysis of the Raman spectra of thymidine showed that the wavenumber of several bands presents jumps at about 160 K, suggesting the occurrence of a conformational modification due change of hydrogen bonds. A comparison with the behavior of amino acid crystals will be welcome, and we hope that in a few time we will have an overview of the subject.
![](http://cdnintech.com/media/chapter/52839/1512345123/media/fig12.png)
Figure 12.
Raman spectrum of thymidine; in the inset a representation of the molecule.
In résumé, in this chapter, a complete picture about the Raman spectra of the 20 proteinogenic amino acid crystals was furnished and some aspects related to the modification of these spectra under extreme conditions were also discussed. As additional information we discussed the Raman spectrum of thymidine, an organic molecule involved in the formation of DNA.
Acknowledgments
The authors acknowledge financial support from CNPq and FUNCAP through PRONEX program.
References
- 1.
Fleck M, Petrosyan AM, Salts of Amino Acids: Crystallization, Structure and Properties. Heidelberg: Springer; 2011. 574 p. DOI: 10.1007/978-3-319-06299-0_1. - 2.
Boldyreva E, Crystalline Amino Acids: A Link between Chemistry, Materials Science and Biology, In: Boyens JCA, Ogilvie JF, editors. Models, Mysteries, and Magic of Molecules. Netherlands: Springer; 2007. pp. 167–192. DOI: 10.1007/978-1-4020-5941-4. - 3.
Freire PTC, Pressure-Induced Phase Transitions in Crystalline Amino Acids, In: Boldyreva E, Dera P, editors, High Pressure Crystallography – From Fundamental Phenomena to Technological Applications. New York: Springer; 2010. pp. 559–572. DOI: 10.1007/978-90-481-9258-8. - 4.
Görbitz CH, Crystal structures of amino acids: from bond lengths in glycine to metal complexes and high-pressure polymorphs, Crystallography Reviews 2015; 21: 160–212, DOI: 10.1080/0889311X.2014.964229. - 5.
Freire PTC, Lima Jr JA, Abagaro BTO, Pinheiro GS, Silva JAF, Filho JM, Melo FEA, High Pressure Raman Spectra of Amino Acid Crystals. In: Dominique de Caro, editor. Vibrational Spectroscopy. Rijeka: InTech; 2012. pp. 37–58. DOI: 10.5772/1345. ch2 - 6.
Boldyreva EV, Drebushchak VA, Drebushchak TN, Paukov IE, Kovalevskaya YA, Shutova ES, Polymorphism of glycine. Thermodynamic aspects. Part I: Relative stability of the polymorphs, Journal of Thermal Analysis and Calorimetry. 2003; 73: 409–418. DOI: 10.1023/A:1025405508035. - 7.
Murli C, Sharma SM, Karmakar S, Sikka SK, α-glycine under high pressures: a Raman study, Physica B 2003; 339: 23–30. DOI: 10.1016/S0921-4526(03)00446-0. - 8.
Goryainov SV, Kolesnik EN, Boldyreva EV, A reversible pressure-induced phase transition in β-glycine at 0.76 GPa, Physica B 2005; 357: 340–357. DOI: 10.1016/j.physb.2004.11.089. - 9.
Boldyreva EV, Drebushchak VA, Drebushchak TN, Paukov IE, Kovalevskaya YA, Shutova ES, Polymorphism of glycine, Part II, Journal of Thermal Analysis and Calorimetry 2003; 73: 419–428. DOI: 10.1023/A:1025457524874. - 10.
Boldyreva EV, Ivashevskaya SN, Sowa H, Ahsbahs H, Weber H-P, Effect of hydrostatic pressure on the γ-polymorph of glycine 1. A polymorphic transition into a new δ-form, Zeitschrift für Kristallographie 2005; 220: 50–57. DOI: 10.1524/zkri.220.1.50.58886. - 11.
Goryainov SV, Boldyreva EV, Kolesnik EM, Raman observation of a new (ζ) polymorph of glycine? Chemical Physics Letters 2006; 419: 406–500. DOI: 10.1016/j.cplett.2005.11.123. - 12.
Wang CH, Storms RD, Raman study of hydrogen bonding and long-wavelength lattice modes in an L-alanine single crystal, Journal of Chemical Physics 1971; 55 : 5110–5119. DOI: 10.1063/1.1675629. - 13.
Susi H, Byler DM, Vibrational analysis of L-alanine and deuterated analogs, Journal of Molecular Structure 1980; 63: 1–11. DOI: 10.1016/0022-2860(80)85305-1. - 14.
Diem M, Polavarapu PL, Oboodi M, Nafie LA, Vibrational circular dichroism in amino acids and peptides. 4. Vibrational analysis, assignments, and solution-phase Raman spectra of deuterated isotopomers of alanine, Journal of American Chemistry Society 1982; 104: 3329–3336. DOI: 10.1021/ja00376a014. - 15.
Rozenberg M, Shoham G, Reva I, Fausto R, Low-temperature Fourier transform infrared spectra and hydrogen bonding in polycrystalline L-alanine, Spectrochimica Acta A 2003; 59: 3253–3266. DOI: 10.1016/S1386-1425(03)00159-8. - 16.
Vik AF, Yuzyuk YI, Barthes M, Sauvajol J-L, Low-wavenumber dynamics of L-alanine, Journal of Raman Spectroscopy 2005; 36: 749–754. DOI: 10.1002/jrs.1328. - 17.
Freire PTC, Melo FEA, Mendes Filho J, Lima RJC, Teixeira AMR, The behavior of NH3 torsional vibration of L-alanine, L-threonine and taurine crystals under high pressure: a Raman spectroscopic stugy, Vibrational Spectroscopy 2007; 45: 99–102. DOI: 10.1016/j.vibspec.2007.05.003. - 18.
Teixeira AMR, Freire PTC, Moreno AJD, Sasaki JM, Ayala AP, Mendes Filho J, Melo FEA, High-pressure Raman study of L-alanine crystal, Solid State Communications 2000; 116: 405–409. DOI: 10.1016/S0038-1098(00)00342-2. - 19.
Tumanov NA, Boldyreva EV, Kolesov BA, Kurnosov AV, Cabrera RQ, Pressure-induced phase transitions in L-alanine, revisited, Acta Crystallographica 2010; B 66: 458–471. DOI: 10.1107/5010876811001983X. - 20.
Funnell NP, Dawson A, Francis D, Lennie AR, Marshall WG, Moggach AS, Warren JE, Parsons S, The effect of pressure on the crystal structure of L-alanine, CrysEngComm 2010; 12: 2573–2583. DOI: 10.1039/c001296c. - 21.
Lima Jr. JA, Freire PTC, Lima RJC, Moreno AJD, Mendes Filho J, Melo FEA, Raman scattering of L-valine crystals, Journal of Raman Spectroscopy 2005; 36: 1076–1081. DOI: 10.1002/jrs.1410. - 22.
Façanha Filho PF, Freire PTC, Lima KVC, Mendes Filho J, Melo FEA, Pizani PS, High temperature Raman spectra of L-leucine crystals, Brazilian Journal of Physics 2008; 38: 131–137. DOI: 10.1590/S0103-97332008000100024. - 23.
Façanha Filho PF, Freire PTC, Melo FEA, Lemos V, Mendes Filho J, Pizani PS, Rossatto DZ, Pressure-induced phase transitions in L-leucine crystals, Journal of Raman Spectroscopy 2009; 40: 46–51. DOI: 10.1002/jrs.2071. - 24.
Almeida FM, Freire PTC, Lima RJC, Remédios CMR, Mendes Filho J, Melo FEA, Raman spectra of L-isoleucine crystals, Journal of Raman Spectroscopy 2006; 37: 1296–1301. DOI: 10.1002/jrs.1553. - 25.
Olsztynska S, Komorowska M, Vrielynck L, Dupuy N, Vibrational spectroscopic study of L-phenylalanine: effect of pH, Applied Spectroscopy 2001; 55: 901–907. DOI: 0003-7028/01/5507-0901. - 26.
Chuang C-H, Chen Y-T, Raman scattering of L-tryptophan enhanced by surface Plasmon of silver nanoparticles: vibrational assignment and structural determination, Journal of Raman Spectroscopy 2009; 40: 150–156. DOI:10.1002/jrs.2097 - 27.
Lima JA, Freire PTC, Melho FEA, Lemos V, Mendes Filho J, Pizani PS, High pressure Raman spectra of L-methionine crystal, Journal of Raman Spectroscopy 2008; 39: 1356–1363. DOI: 10.1002/jrs.2005 - 28.
Kolesov BA, Minkov VS, Boldyreva EV, Drebushchak TN. Phase transitions in the crystals of L- and DL-cysteine on cooling: intermolecular hydrogen bonds distortions and the side-chain motions of thiol-groups. 1. L-cysteine, Journal of Physical Chemistry B 2008; 112: 12827–12839. DOI: 10.1021/jp804142c. - 29.
Barthes M, Bordallo HN, Dénoyer F, Lorenzo J-E, Zaccaro J, Robert A, Zontone F. Micro-transitions or breathers in L-alanine? The European Physical Journal B 2004; 37: 375–382. DOI: 10.1140/epjb/e2004-00069-1. - 30.
Souza JM, Freire PTC, Bordallo HN, Argyriou DN. Structural isotopic effects in the smallest chiral amino acid: observation of a structural phase transition in fully deuterated alanine. Journal of Physical Chemistry B 2007; 111: 5034–5039. DOI: 10.1021/jp070366z. - 31.
Minkov VS, Krylov AS, Boldyreva EV, Goryainov SV, Bizyaev SN Vtyurin AN, Pressure-induced phase transitions in crystalline L- and DL-cysteine, Journal of Physical Chemistry B 2008; 112: 8851–8854. DOI: 13.1021/jp8020276. - 32.
Kolesov BA, Boldyreva EV, Difference in the dynamic properties of chiral and racemic crystals of serine studied by Raman spectroscopy at 3–295 K, Journal of Physical Chemistry B 2007; 111: 14387–14397. DOI: 10.1021/jp076083o. - 33.
Dhamelincourt P, Famirez FJ, Polarized micro-Raman and FT-IR spectra of L-glutamine, Applied Spectroscopy 1993; 47: 446–451. DOI: 10.1366/0003702934335083. - 34.
Moreno AJD, Freire PTC, Melo FEA, Mendes Filho J, Nogueira MAM, Almeida MA, Miranda MAR, Remédios CMR, Sasaki JM, Low-temperature Raman spectra of monohydrated L-asparagine crystals, Journal of Raman Spectroscopy 2004; 35: 236–241. DOI: 10.1002/jrs.1141. - 35.
Bento ICV, Freire PTC, Melo FEA, Mendes Filho J, Moreno AJC, Joya MR, Pizani PS, High temperature phase transition in monohydrated L-asparagine crystal, Solid State Communications 2007; 141: 29–32. DOI: 10.1016/j.ssc.2006.09.041. - 36.
Silva JAF, Freire PTC, Lima JA, Mendes Filho J, Melo FEA, Moreno AJD, Polian A, Raman spectroscopy of monohydrated L-asparagine up to 30 GPa, Vibrational Spectroscopy 2015; 77: 35–39. DOI: 10.1016/j.vibspec.2015.02.006. - 37.
Silva BL, Freire PTC, Guedes I, Araújo-Silva MA, Mendes Filho J, Moreno AJD, Polarized Raman spectra and infrared analysis of vibrational modes in L-threonine crystals, Brazilian Journal of Physics 1998; 28: 19–24. DOI: 10.1590/S0103-97331998000100003. - 38.
Holanda RO, Lima JA, Freire PTC, Melo FEA, Mendes Filho J, Polian A, New pressure-induced phase transitions of L-threonine crystal: a Raman spectroscopic study, Journal of Molecular Structure 2015; 1092: 160–165. DOI: 10.1016/j.molstruc.2015.03.024. - 39.
Luz-Lima C, Sousa GP, Lima Jr. JA, Melo FEA, Mendes Filho J, Polian A, Freire PTC, High pressure Raman spectra of β form of L-glutamic acid, Vibrational Spectroscopy 2012; 48: 181–187. DOI: 10.1016/j.vibspec.2011.12.005. - 40.
Hernández B, Pflüger F, Derbel N, De Coninck J, Ghomi M, Vibrational analysis of amino acids and short peptides in hydrated media. VI. Amino acids with positively charged side chains: L-Lysine and L-Arginine, Journal of Physical Chemistry B 2010; 114: 1077–1088. DOI: 10.1021/jp909517r. - 41.
De Sousa GP, Freire PTC, Mendes Filho J, Melo FEA, Lima CL, Low-temperature Raman spectra of L-histidine crystals, Brazilian Journal of Physics 2013; 43: 137–144. DOI: 10.1007/s13538-013-0132-3. - 42.
Faria JLB, Almeida FM, Pilla O, Rossi F, Sasaki JM, Melo FEA, Mendes Filho J, Freire PTC, Raman spectra of L-histidine hydrochloride monohydrate crystal, Journal of Raman Spectroscopy 2004; 35: 242–248. DOI: 10.1002/jrs.1142. - 43.
Long DA, The Raman Effect, John Wiley & Sons, Chichester 2002. DOI: 10.1002/0470845767. - 44.
Salam A, Chirality, phase transitions and their induction in amino acids, Physics Letters B 1992; 288: 153–160. DOI: 10.1016/0370-2693(92)91970-K.