**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 lasparagine.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.

It is important to remember that *l-cysteine* can be found without HCl ions in the unit cell. lcysteine can be obtained under ambient conditions in two different crystalline polymorphs, orthorhombic, and monoclinic. The orthorhombic structure of l-cysteine crystallizes with *Z* = 4 and space group *P*212121. One of the important characteristics among the amino acid crystals is the fact that l-cysteine presents the S-H…S hydrogen bond, extending along the **b** crystallographic direction. As a consequence, l-cysteine constitutes a model to understand the important sulfur hydrogen bonds involved in some proteins of the human being. In a polarized Raman spectroscopic study, it was observed that at low temperatures, the S-H…S hydrogen bonds contribute to form an ordered crystal structure, but upon heating, the thiolgroups appear slightly disordered [28]. As pointed out in this reference, some of the S-H…S hydrogen bonds are substituted by S-H…O bonds in such a way that at room temperature the number of the two species of hydrogen bonds is approximately the same. Interestingly enough is the fact that the change of hydrogen bonds with the substitution of sulfur by oxygen is not sharp, but occurs through a series of intermediate states [28]. We remember that lalanine, when submitted to low temperature conditions, also presents a pathological behavior, i.e., the **c** crystallographic parameter decreases in a succession of steps and plateaus. The jumps on the **c** parameter were interpreted for l-alanine as attempts to relax some frustration [29]. Returning to the l-cysteine case, the modification in the thiol group is related to different orientations of the cysteine zwitterion, which are tuned by the strong N-H…O hydrogen bonds. Additionally, it was observed that different groups (NH3, SH, CH, and CH2) are activated in different temperature ranges, similarly with was observed for the NH3 and CH3 groups of l-alanine [30]. Under high pressure, the Raman spectrum of orthorhombic lcysteine presents noticeable modifications that can be summarized as follows [31]. Above 0.1 GPa redistribution of intensities of the components of the bands associated with stretching of SH, ν(SH), suggests a continuous decrease in the number of sulfhydryl groups participating of S-H…S hydrogen bonds. This tendency remains until the pressure arrive to 1.6–1.9 GPa, when is observed an impressive change in the Raman spectrum, associated with a phase transition. One of these changes is the downshift of the wavenumber of ν(SH) by ~ 40 cm−1, as well as the splitting of this band; such facts suggest both (i) the S-H…S hydrogen bond

l-methionine [27]. In relation to the previous amino acid presented in this chapter, phenylalanine and tryptophan show an additional benzene ring. As a consequence, this unit presents vibrations associated with it: stretching of CH at 2979, 3030, and 3057 cm−1; rocking at ~1260 cm−1; deformation at about 704 cm−1; ring breathing at ~ 1010 cm−1 and wagging at ~ 744 cm−1, for l-tryptophan [25]. The l-phenylalanine shows that vibrations related to the benzene ring are observed at 848, 912, and 949 cm−1 (out-of-plane bending); 1001 cm−1 (breathing of the ring); 1025 and 1076 cm−1 (in-plane bending); 1600 cm−1 (stretching of CC in the ring). Some results on l-proline monohydrated were presented in reference [5], where a high pressure investigation is furnished; the work shows evidence of two phase transitions, between 0.0 and 1.1 GPa and another between 6.5 and 7.8 GPa. In relation to lmethionine, a detailed study of the Raman spectra of the crystal under high pressure was revealed in reference [27]. Although the methionine molecule presents a sulfur atom, differently from the cysteine, in the methionine there is a connection linking two carbon atoms, C–S–C; as a consequence, the very intense band observed at ~ 2500 cm−1 appearing in the Raman spectrum of cysteine due to the S-H stretching is not observed in the Raman spectrum of methionine. However, a very characteristic band associated with the C-S stretching vibration is observed at 659 cm−1 in the spectrum of l-methionine. Following this band, when the l-methionine crystal was subjected to high pressure into a diamond anvil cell, it was observed an impressive modification that was associated with a phase transition undergone by the crystal at about 2.2 GPa [27]. Also interesting is the fact that the phase transition occurs with a hysteresis of ~ 0.8 GPa, suggesting the transition can be classified

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 lasparagine.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. It is important to remember that *l-cysteine* can be found without HCl ions in the unit cell. lcysteine can be obtained under ambient conditions in two different crystalline polymorphs, orthorhombic, and monoclinic. The orthorhombic structure of l-cysteine crystallizes with *Z* = 4 and space group *P*212121. One of the important characteristics among the amino acid crystals is the fact that l-cysteine presents the S-H…S hydrogen bond, extending along the **b** crystallographic direction. As a consequence, l-cysteine constitutes a model to understand the important sulfur hydrogen bonds involved in some proteins of the human being. In a polarized Raman spectroscopic study, it was observed that at low temperatures, the S-H…S hydrogen bonds contribute to form an ordered crystal structure, but upon heating, the thiolgroups appear slightly disordered [28]. As pointed out in this reference, some of the S-H…S

as a first-order one.

210 Raman Spectroscopy and Applications

**4. Neutral, polar amino acids**

**Figure 7.** FT-Raman spectra of polar (neutral) amino acids l-serine, l-glutamine and compounds l-cysteine.HCl and lasparagine.H2O. In the spectrum of l-cysteine.HCl stands out an intense band associated with the S-H stretching, ν(SH), similar to the monoclinic phase of l-cysteine, but different from the orthorhombic phase, where broad split bands are observed (see text).

strengthen and (ii) increasing of disorder of SH group. Above 2.3 GPa, the wavenumber of ν(SH) presents a upshift of 30 cm−1, indicating the weakening of hydrogen bonds related to S-H groups. However, the modes are split above this pressure, and we can consider that the sulfhydryl groups are still disordered.

Obviously, if you change the sulfur atom by an oxygen atom, the hydrogen bonds involving SH groups cease to exist. As a molecule, *serine* is a copy of cysteine but with oxygen replacing the sulfur atom. l-serine crystallizes in an orthorhombic structure with space group *P*212121, the same of one of the polymorphs of l-cysteine. A Raman spectroscopic study showed that l-serine presents changes at ~ 140 K. the changes were interpreted as reorientation of the side chain CH2OH with respect to the C-C bonds of the skeleton of the molecule [32]. As a consequence, a positional disorder of the O-H...O intermolecular hydrogen bond is verified. Such a fact was realized through the analysis of the behavior of stretching of OH, ν(OH), allowing to separate the temperature evolution of O-H...O hydrogen bond among the other formed by serine molecules in the crystal structure: N-H...O in the head-to-tail chains, N-H...O between antiparallel chains and N-H...O between **ab** layers [32]. This constitutes a very beautiful example of the power of Raman spectroscopy to play light in a so complicated theme as is hydrogen bond.

*Glutamine* is the more abundant proteinogenic amino acid in the human blood, occupying a pivotal position in the nitrogen metabolism. The radical of the amino acid is characterized by the groups NH2-(C=O)-CH2-CH2. As a consequence, the high spectral region of the Raman spectrum of l-glutamine presents a rich profile. In the FT-Raman spectrum a very strong band observed at 2933 cm−1 is associated with the symmetric stretching of CH2, νS(CH2); a doublet at 2952 and 2962 cm−1 is associated, respectively, with the stretching CH, ν(CH), and the symmetric stretching of CH2, νS(CH2); a peak observed at 2991 cm−1 is assigned as antisymmetric stretching of CH2, νA(CH2). Above 3100 cm−1 it is possible to observe bands with low intensity associated with NH2 and NH3 + groups: at 3176 cm−1, assigned as symmetric stretching of NH2; at 3210 cm−1, assigned as symmetric stretching of NH3 + and at 3403 cm−1, assigned as antisymmetric stretching of NH2. Such assignment, performed on reference [33] with the use of deuterated l-glutamine samples will be fundamental to understand the behavior of the crystal submitted to extreme conditions e.g., high pressure and low temperatures.

The radical NH2-(C=O)-CH2 characterizes the amino acid l-*asparagine*. Although is possible to grow small crystals of the pure form, most of the studies on vibrational spectroscopy deals with the hydrate form, monohydrated l-asparagine (MLA). This crystal was studied in a series of papers [34, 35]. A Raman spectroscopic study revealed that under low temperature MLA undergoes a phase transition between 140 and 150 K. The modification is clearly realized through the observation of splitting of a band assigned as lattice modes, at ~ 130 cm−1 [34]. Under high temperature, MLA also presents a phase transition, as it was shown by Raman scattering measurements [35]. At about 363 K, the orthorhombic *P*212121 structure of MLA change drastically, as it is possible to infer from the impressive modifications of the Raman spectra above this temperature. Interesting enough, while in the low temperature phase transition the modifications are observed mainly in the low wavenumber region of the Raman spectrum, in the high temperature phase transition modifications occur in all spectral range. Something that also deserves attention is the behavior of MLA under high pressure. In a recent work [36] authors have investigated the material up to 30 GPa. This is the highest pressure value utilized in experiments on the vibrational properties of amino acid crystals up to now (we remember that l-alanine presents a crystal-amorphous phase transition at 15 GPa, half of the pressure value reached in the experiment with MLA). In this work, analyzing most of the Raman bands in the spectral range 30–3600 cm−1, it was possible to note modifications that were correlated with phase transition undergone by MLA, as well as, with conformational changes of the molecules in the unit cell of the crystal. The changes observed at approximately 10 GPa were associated with a phase transition and other modifications between 2.1 and 3.1 GPa and between 15.0 and 17.0 GPa were associated with conformational changes. In particular, the most impressive modifications occur in the high wavenumber region of the spectrum. Very suggestive is the fact that the wavenumbers of the antisymmetric stretching of NH2, νA(NH2) and symmetric stretching of H2O, νS(H2O) decrease in the interval at 1 atm and 8.5 GPa, indicating that hydrogen bonds are strengthened in this pressure interval. The explanation for the anomalous behavior is because N-H…O hydrogen bond interaction is stiffened due the approximation of molecules under compression, weakening the covalent N-H interaction and, consequently, shifting the wavenumber of the two modes to lower values. During the transition at 10 GPa, the bands associated with stretching of water molecule show a positive jump, indicating a new environment for the molecules. However, between 10.6 and 15 GPa and above 17.9 GPa the symmetric stretching of water goes, respectively, to higher and to lower wavenumbers, signaling different behavior of the hydrogen bonds. As a résumé for the data on MLA, we can affirm that Raman spectroscopy furnished a precise picture about the hydrogen bonds allocated in the unit cell of the crystal.

strengthen and (ii) increasing of disorder of SH group. Above 2.3 GPa, the wavenumber of ν(SH) presents a upshift of 30 cm−1, indicating the weakening of hydrogen bonds related to S-H groups. However, the modes are split above this pressure, and we can consider that the

Obviously, if you change the sulfur atom by an oxygen atom, the hydrogen bonds involving SH groups cease to exist. As a molecule, *serine* is a copy of cysteine but with oxygen replacing the sulfur atom. l-serine crystallizes in an orthorhombic structure with space group *P*212121, the same of one of the polymorphs of l-cysteine. A Raman spectroscopic study showed that l-serine presents changes at ~ 140 K. the changes were interpreted as reorientation of the side chain CH2OH with respect to the C-C bonds of the skeleton of the molecule [32]. As a consequence, a positional disorder of the O-H...O intermolecular hydrogen bond is verified. Such a fact was realized through the analysis of the behavior of stretching of OH, ν(OH), allowing to separate the temperature evolution of O-H...O hydrogen bond among the other formed by serine molecules in the crystal structure: N-H...O in the head-to-tail chains, N-H...O between antiparallel chains and N-H...O between **ab** layers [32]. This constitutes a very beautiful example of the power of Raman spectroscopy to play light in a so complicated theme as is

*Glutamine* is the more abundant proteinogenic amino acid in the human blood, occupying a pivotal position in the nitrogen metabolism. The radical of the amino acid is characterized by the groups NH2-(C=O)-CH2-CH2. As a consequence, the high spectral region of the Raman spectrum of l-glutamine presents a rich profile. In the FT-Raman spectrum a very strong band observed at 2933 cm−1 is associated with the symmetric stretching of CH2, νS(CH2); a doublet at 2952 and 2962 cm−1 is associated, respectively, with the stretching CH, ν(CH), and the symmetric stretching of CH2, νS(CH2); a peak observed at 2991 cm−1 is assigned as antisymmetric stretching of CH2, νA(CH2). Above 3100 cm−1 it is possible to observe bands with low

antisymmetric stretching of NH2. Such assignment, performed on reference [33] with the use of deuterated l-glutamine samples will be fundamental to understand the behavior of the

The radical NH2-(C=O)-CH2 characterizes the amino acid l-*asparagine*. Although is possible to grow small crystals of the pure form, most of the studies on vibrational spectroscopy deals with the hydrate form, monohydrated l-asparagine (MLA). This crystal was studied in a series of papers [34, 35]. A Raman spectroscopic study revealed that under low temperature MLA undergoes a phase transition between 140 and 150 K. The modification is clearly realized through the observation of splitting of a band assigned as lattice modes, at ~ 130 cm−1 [34]. Under high temperature, MLA also presents a phase transition, as it was shown by Raman scattering measurements [35]. At about 363 K, the orthorhombic *P*212121 structure of MLA change drastically, as it is possible to infer from the impressive modifications of the Raman spectra above this temperature. Interesting enough, while in the low temperature phase transition the modifications are observed mainly in the low wavenumber region of the Raman spectrum, in the high temperature phase transition modifications occur in all spectral range.

groups: at 3176 cm−1, assigned as symmetric stretching

and at 3403 cm−1, assigned as

+

+

crystal submitted to extreme conditions e.g., high pressure and low temperatures.

of NH2; at 3210 cm−1, assigned as symmetric stretching of NH3

sulfhydryl groups are still disordered.

212 Raman Spectroscopy and Applications

intensity associated with NH2 and NH3

hydrogen bond.

**Figure 8.** FT-Raman spectrum of l-tyrosine. It is interesting to observe the intense peaks characterizing the low wavenumber of the spectrum. The inset presents a representation of the molecule.

The FT-Raman spectrum of *l-tyrosine* is shown in **Figure 8** (the inset shows a representation of the molecular formula). As occurs with l-phenylalanine and l-tryptophan, l-tyrosine 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 pressure conditions, among other extreme conditions.

**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 threonine is a relatively simple molecule.

**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 907–940 cm−1. Rocking of CO2 − , r(CO2 − ) is observed at ~ 568 cm−1. It is interesting in this point remember that r(CO2 − ) vibration is observed at 515 cm−1 in l-serine, 530 cm−1 in l-histidine 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 it is observed. It is also relevant to point out that the torsion of NH3 + , *τ*(NH3 + ), is observed in l-threonine at 497 cm−1. A recent Raman scattering study—where a sample of l-threonine was put into a diamond anvil cell set up—showed an impressive phase transition close to 2 GPa, another between 8.2 and 9.2 GPa, and a third between 14 and 15.5 GPa. The principal indication for the occurrence of these pressure-induced phase transitions is the modification in the bands associated with external modes. Additional changes observed in bands related to CO2, NH3, and CH3 units of the threonine molecule also corroborate the occurrence of the three-phase transition [38]. However, although the maximum pressure reached in the experiment was 27 GPa, no evidence of amorphization was observed. This point is being analyzed because, on the contrary, l-alanine undergoes a crystal — amorphous phase transition for pressure of only 15 GPa. Additionally, a comparative study looking for a correlation between the behavior of NH3 torsional modes of l-alanine, l-threonine, and taurine with the hydrogen bond dimensions was given in reference [17]. A possible connection between the hydrogen bond dimensions and the amorphous state of the amino acid crystal can give interesting insights about the phenomenon.
