**5. Acidic, polar amino acids**

presents a benzene ring in its radical. Consequently, it is expected that vibrations associated with breathing of the benzene ring and CC stretching related to the atoms of the ring appear in the Raman spectrum of l-tyrosine with frequencies similar to those of l-phenylalanine and l-tryptophan. It is also interesting to observe that the low wavenumber bands are the most intense bands appearing in the Raman spectrum. Because these bands are assigned mainly as lattice modes, it will be of interest in future studies where one should search for eventual phase transitions. In the high wavenumber region of the Raman spectrum, it is possible to observe distinctly five different bands that are associated with the stretching vibrations of CH and CH2, ν(CH) and ν(CH2), respectively. Up to now, there is no work published in the literature discussing the vibrational behavior of l-tyrosine subjected to neither low temperature nor high

**Figure 9.** FT-Raman spectrum of l-threonine in the spectral range 20–3500 cm−1 recorded at room temperature. It is interesting the observation of a very complex profile in the high wavenumber region of the spectrum although threo-

**Figure 9** shows the FT-Raman spectrum of *l-threonine* in the spectral range from 20 to 3500 cm−1. Most of the bands appearing in the low wavenumber region are associated with lattice modes and, as a consequence, their behavior can furnish information about the stability of the unit cell [37]. l-threonine crystallizes in an orthorhombic structure with space group *P*212121. Vibrations associated with CC stretching are observed as bands in the spectral range

hydrochloride monohydrated, 553 cm−1 in l-asparagine monohydrated and in l-cysteine, 545 cm−1 in l-methionine and 541 cm−1 in l-valine. So, this kind of vibration that appears in the Raman spectrum as a band of relatively high intensity presents a well-specific range where

) is observed at ~ 568 cm−1. It is interesting in this point

) vibration is observed at 515 cm−1 in l-serine, 530 cm−1 in l-histidine

pressure conditions, among other extreme conditions.

214 Raman Spectroscopy and Applications

nine is a relatively simple molecule.

907–940 cm−1. Rocking of CO2

−

remember that r(CO2

− , r(CO2 − **Figure 10** shows the FT-Raman spectra of *l-aspartic acid* and *l-glutamic acid* in the spectral range from 20 to 3500 cm−1. In some aspects, the two spectra are similar, e.g., the lattice modes appear as very intense bands in the low wavenumber region of the spectrum and the profiles of the Raman spectra in the high wavenumber region are very similar for the two crystals. l-glutamic acid crystallizes in two possible polymorphs, called α (like prisms) and β (as platelets). Both polymorphs crystallize in an orthorhombic structure in a *P*212121 space group. In **Figure 10**, the main contribution for the FT-Raman spectrum of l-glutamic acid is from the β-form. For this polymorph, there is no intramolecular hydrogen bond, but a strong hydrogen bond between two carboxylic groups in neighboring molecules is observed; such a bond form links along the *b*-direction. In the l-glutamic acid, the symmetric stretching of NH3 + appears— as occurs with most of the amino acid crystals—as a weak band, observed at 3073 cm−1. A very strong band associated with the stretching of CH2 is observed at 2974 and 2938 cm−1. Vibrations associated with rocking of NH3 + are observed at 1128 and 1149 cm −1 and the stretching of CC is observed at 970 and 1062 cm−1. At 804 and 866 cm−1 (the last one as a very strong band) bands are associated with rocking of CH2. Vibrations associated with the skeleton of the l-glutamic acid are observed in the spectral range of 240–398 cm−1. Finally, the torsion of CO2 − unit is observed at 199 cm−1 and bands with wavenumber lower than this value are associated with the lattice vibrations of the crystal. Raman spectroscopy was used to investigate the vibrational properties of a crystal of l-glutamic acid in its β−form. From this study, authors have observed modifications that can be considered as evidence that the crystal undergoes some phase transitions. One modification in the Raman spectrum was observed between 0.5 and 1.3 GPa. The second modification was noted between 2.6 and 3.1 GPa; the third modification was observed for pressures between 5.4 and 6.4 GPa and the fourth change in the Raman spectra was observed for pressures between 13.9 and 15.9. Again, it is important to inform that at these pressures there is no evidence of amorphization for the crystal of l-glutamic acid; in fact, even at the highest pressure reached in the experiments (21.5 GPa), the crystal shows all bands, given no evidence of an amorphous phase [39].

**Figure 10.** FT-Raman spectra of l-aspartic acid and l-glutamic acid in the spectral range 20–3500 cm-1 recorded at room temperature.

#### **6. Basic, polar amino acids**

*l-Lysine*, *l-arginine* and *l-histidine* are the basic, polar amino acids. Lysine has as characteristic a radical composed of the following group of atoms CH2–CH2–CH2–CH2–NH2. Almost no work on Raman spectroscopy of l-lysine was published. However, Hernández et al. [40] showed the assignment of the main modes of the Raman spectrum of l-lysine. For example, the stretching of CC is observed at 1012, 1033, 1063, and 1076 cm−1; the antisymmetric rocking of NH3 + is observed at 1143 and 1183 cm−1; the antisymmetric bending of NH3 + appears as bands at 1615 and 1650 cm−1; the symmetric stretching of CO2 − at 1415 cm−1 and the antisymmetric stretching of CO2 − at 1598 cm−1. Hernández et al. [40] also presented a tentative assignment of most bands appearing in the Raman spectrum of l-arginine. The radical characterizing l-arginine is CH2–CH2–CH2–NH–C–NH–NH2. The Raman spectrum of larginine shows bands at 1011 and 1035 cm−1 which are associated with CC stretching, while a band at 970 cm−1 is associated with CN stretching. Rocking of NH3 + is observed at 1164 cm −1 and antisymmetric bending of CNH2 is observed at 1176 cm−1. The symmetric stretching of CO2 − is observed at 1581 cm−1 and the antisymmetric stretching of CO2 − appears at 1581 cm−1 [40].

it is important to inform that at these pressures there is no evidence of amorphization for the crystal of l-glutamic acid; in fact, even at the highest pressure reached in the experiments (21.5 GPa), the crystal shows all bands, given no evidence of an amorphous phase [39].

**Figure 10.** FT-Raman spectra of l-aspartic acid and l-glutamic acid in the spectral range 20–3500 cm-1 recorded at room

*l-Lysine*, *l-arginine* and *l-histidine* are the basic, polar amino acids. Lysine has as characteristic a radical composed of the following group of atoms CH2–CH2–CH2–CH2–NH2. Almost no work on Raman spectroscopy of l-lysine was published. However, Hernández et al. [40] showed the assignment of the main modes of the Raman spectrum of l-lysine. For example, the stretching of CC is observed at 1012, 1033, 1063, and 1076 cm−1; the antisymmetric rocking

is observed at 1143 and 1183 cm−1; the antisymmetric bending of NH3

assignment of most bands appearing in the Raman spectrum of l-arginine. The radical characterizing l-arginine is CH2–CH2–CH2–NH–C–NH–NH2. The Raman spectrum of larginine shows bands at 1011 and 1035 cm−1 which are associated with CC stretching, while

−

+

at 1598 cm−1. Hernández et al. [40] also presented a tentative

bands at 1615 and 1650 cm−1; the symmetric stretching of CO2

a band at 970 cm−1 is associated with CN stretching. Rocking of NH3

−

+

is observed at 1164 cm

at 1415 cm−1 and the antisym-

appears as

temperature.

of NH3 +

**6. Basic, polar amino acids**

216 Raman Spectroscopy and Applications

metric stretching of CO2

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 *P*212121 space group with four molecules per unit cell. It is interesting to note that many of the amino acids crystallize in a *P*212121 space group with orthorhombic structure or in a *P*21 space group with monoclinic structure; this possibly is related to the packing of molecules in the unit cell, but this point will not be explored in the present text. Returning to the case of l-histidine, the Raman scattering study in reference [41] showed a series of discontinuity in the wavenumber of bands at about 165 K. This was interpreted as consequence of a conformational phase transition through involving both CO2 − and NH3 + groups. It is interesting to add the information that l-arginine and l-histidine can also easily grow as hydrated and as chloride hydrated crystals. In **Figure 11**, the Raman spectra of l-histidine hydrochloride monohydrated crystal are shown for three different scattering geometries in order to illustrate a case of an amino acid crystallizing with water and HCl units.

**Figure 11.** Raman spectra of l-histidine hydrochloride monohydrate in three scattering geometries (adapted from reference [43]).

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.

**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.
