Vibrational Spectroscopy 89 (2017) 62–68

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Vibrational Spectroscopy
journal homepage: www.elsevier.com/locate/vibspec

High-pressure Raman spectra of thymidine crystals

F.M. Barbozaa , J.G. da Silva Filhoa , P.T.C. Freirea,* , P.F. Façanha Filhob , J.A. Lima Jr.a ,
F.E.A. Meloa , M.R. Joyac , J. Barba-Ortegac
a
Departamento de Física, Universidade Federal do Ceará, C.P. 6030, Campus do Pici, 60455-760, Fortaleza, CE, Brazil
b
Centro de Ciências Sociais, Saúde e Tecnologia (CCSST) Universidade Federal do Maranhão – Campus II, 65900-000, Imperatriz, MA, Brazil
c
Universidad Nacional de Colombia– Bogotá, Facultad de Ciências, Departamento de Física, Carrera 30 Calle 45-03, C.P. 111321, Bogotá, Colombia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 6 November 2016 Raman spectra of thymidine crystal were obtained for pressures up to 5.0 GPa in a diamond anvil cell. The
Accepted 16 December 2016 results show the presence of anomaly in the Raman spectrum at pressures close to 3.0 GPa. This anomaly
Available online 21 January 2017 is characterized by disappearance of lattice modes, appearance of some internal modes, splitting of high
wavenumbers modes, downshift of modes associated with hydrogen bonds, changes in the intensity of
Keywords: internal modes and discontinuities of the slopes of the wavenumbers versus pressure for several Raman
Nucleosides modes. This set of modifications was interpreted as consequence of a phase transition undergone by
Hydrogen bonds thymidine close to 3.0 GPa. Further, decompression to atmospheric pressure generates the original
High pressure
Raman spectrum, showing that the pressure-induced phase transition undergone by thymidine crystals
is reversible. A comparison with results on other nucleosides submitted to high pressure is also furnished.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
The nucleic acids DNA and RNA are long molecules made up of a
base, a sugar (deoxyribose for DNA and ribose for RNA) and a
phosphate group [1]. In the crystalline form these molecules
constitute very special systems in which hydrogen bonds (HB’s)
between bases are greatly responsible for the stability of double-
helical array. The bases of DNA and RNA are heterocyclic (carbon-
and nitrogen- containing) aromatic rings, with a variety of
substituents. Adenine (A) and guanine (G) are purines, meaning
that their structures are two fused carbon-nitrogen rings, whereas
cytosine (C), thymine (T) and uracil (U) are pyrimidines having only
a single carbon-nitrogen ring. In RNA, the thymine base is replaced
by uracil [2]. The unit of sugar and base connected by a b-glycosyl
linkage is named a nucleoside. If one of the free sugar hydroxyls is
phosphorylated, we have a nucleotide.
Crystals of nucleosides have been studied and no convincing
evidence was furnished pointing to the existence of any structural
phase transition at low temperatures [3–6]. However, under
higher-pressure conditions evidences of phase transitions were
found. For example, adenosine was investigated under high-
pressure, and the study showed that the original monoclinic
* Corresponding author.
E-mail address: tarso@fisica.ufc.br (P.T.C. Freire).
crystal undergoes a phase transition close to 2.5 GPa [7]. It was
mentioned that the modes identified with glycosidic bond down in
frequency near this pressure, which was attributed to a weakening
of the associated bond. Also, the modes associated with hydrogen-
stretching vibrations were found to decrease in frequency with
pressure. It was attributed to a movement of charge from the
molecular bonds involving the associated hydrogen bonds.
Vibrational properties of cytidine crystal were also investigated
for higher pressures. Li et al. [8] reported that this crystal presents a
series of anomalies near 4.0 GPa, which was associated with a
phase transition and attributed to a change in the conformation of
the components of ribose sugar ring.
In this work we studied the vibrational properties of thymidine,
a nucleoside of DNA, which is composed of the deoxyribose sugar
with the pyrimidine base (thymine). The molecular structure and a
view of the unit cell are shown in Figs. 1(a) and (b), respectively.
Hydrogen bonds (HBs) are represented by dashed lines. Extensive
HBs are found in the crystal of thymidine. Taking into account the
biological interest, N3 and O4 are involved in hydrogen bonding,
since they participate in the base pairing of DNA.
Exception for high-pressure and temperature-dependence
Raman experiments, there have been some studies on the
vibrational analysis on thymidine. These studies are directed to
obtain, at room temperature, the dependence of thymidine Raman
spectra on solvent [9], the correct assignment of the internal

http://dx.doi.org/10.1016/j.vibspec.2016.12.013
0924-2031/© 2017 Elsevier B.V. All rights reserved.
 F.M. Barboza et al. / Vibrational Spectroscopy 89 (2017) 62–68 63

Fig. 1. (a) Molecular structure of thymidine; (b) unit cell of thymidine crystal.

vibrations in powder samples [10,11] and to observe vibrational 2. Experimental details

modes by polarized Raman scattering in single crystals [12].
In order to add knowledge and access the structural and
vibrational properties of nucleosides, we performed high-pressure
Raman experiments in thymidine crystal for pressures up to
5.0 GPa. The behavior of several internal modes as well as of
external modes led us to believe in the occurrence of a phase
transition undergone by thymidine crystal in the interval 2.5–
3.0 GPa.
Single crystals of thymidine were grown by slow evaporation
method of an aqueous solution of a commercial reagent (Sigma
Aldrich) at room temperature. It was needed about 2–3 weeks to
obtain crystals in the form of thin elongated colorless plates of
various sizes. The crystal structure of the samples was determined
through an X-ray powder diffraction experiment using a D8
Advance (Bruker) diffractometer using Cu K radiation operating at
40kV–40 mA. The Raman spectra were recorded with a Jobin Yvon

Fig. 2. (a) Raman spectra of thymidine crystals for selected pressures in the spectral region 1730–1350 cm
1; (b) wavenumber versus pressure plot for the spectral region
1730–1350 cm
1.
64 F.M. Barboza et al. / Vibrational Spectroscopy 89 (2017) 62–68
T64000 spectrometer (spectral resolution of  2 cm
1), equipped
with an N2 cooled charge coupled device (CCD) detection system.
The 514 nm line of an argon ion laser was utilized as excitation
source. High-pressure conditions, between 1 atm and 5.0 GPa, was
obtained by standard diamond-anvil cell (DAC) loaded with
mineral oil, which was chosen as compression medium to avoid
interactions such as occurs in the conventional mixture of ethanol-
methanol used as transmitting pressure medium with thymidine
sample. The pressure in the cell was monitored using the shifts of
the ruby emission lines.
3. Results and discussion
The structure of thymidine has been studied by X-ray
diffraction at room temperature [13]. The measurements showed
that this nucleoside crystallizes in the orthorhombic structure
belonging to the P212121 space group. There are four molecules in
the unit cell. Since thymidine has 31 atoms, there will be 87 normal
modes for isolated molecules. In the crystal this number is
quadrupled, resulting from coupling of the motions of the four
molecules. In addition, there are 12 external modes resulting from
molecular librations (hindered rotations) and 12 from translations,
of which three are acoustic. Hence a total of 369 optical modes are
predicted. In terms of the irreducible representations of D2 factor
group, the normal modes are decomposed as G = 93A + 93B1 + 93
B2 + 93B3, and because there are three acoustic modes (G ac = B1 +
B2 + B3), the optical modes are G op = 93A + 92B1 + 92B2 + 92B3,
where all irreducible representations are Raman active and the
B1, B2 and B3 irreducible representations are infrared active.
Typical Raman spectra for several pressure values are shown in
Figs. 2–5 . These spectra show a division between the low-
wavenumber region (below 250 cm
1) and the high-wavenumber
region (above about 250 cm
1). There are some irregularities in the
wavenumber versus pressure plots. It is possible to observe that
the most drastic abnormalities are found near 3.0 GPa. The specific
changes and the evidence they provide for a phase transition near
3.0 GPa will be discussed below.
Before we ponder about the influence of pressure on the
vibrational modes of thymidine crystal we remember that the
structure is linked by a network of hydrogen bonds. As established
Fig. 3. (a) Raman spectra of thymidine crystals for selected pressures in the spectral region 1300–700 cm
1; (b)wavenumber versus pressure plot for the spectral region
1300–700 cm
1.
by the work of Ref. [13] there is a hydrogen bond linking atom O(30 )
and atom O(4) in a neighboring molecule, with an OH  O length of
2.74 Å. Interestingly, the atom O(30 ) accepts a hydrogen atom from
N(3), producing a NH  O hydrogen bond with 2.85 Å. The third
different hydrogen bond occurring in the unit cell of thymidine is
formed between O(50 ) atom from one molecule and the O(50 ) atom
of another molecule, constituting a OH  O bond with 2.79 Å. From
this picture we can expect that the hydrogen bonds have influence
on certain vibrations observed in the Raman spectrum of the
crystal and this can clearly be noted as described in the next
paragraph.
Fig. 2(a) shows the Raman spectra of thymidine crystal in the
spectral range 1730–1350 cm
1 for pressure from 1 atm to 5.0 GPa.
Some aspects of the Raman spectra can be pointed out when
pressure is increased from 1 atm to 2.5 GPa. The first one is a shift
toward low wavenumbers (downshift) which is observed for the
strong band marked by I (at 1664 cm
1), which is associated with
an in-phase stretching motion of C4 = O and C5 = C6 atoms [v
(C4 = O) + v(C5 = C6)]ip [10]. Such a decrease in the wavenumber
with increasing pressure can be evidence that the associated atoms
are involved in a hydrogen bond. In fact, as described in the last
paragraph, the C4 atom is linked to an oxygen atom that
participates of the smallest hydrogen bond in the unit cell of
the thymidine. It is expected that with increasing pressure the
atoms and molecules in the unit cell become closer one to the
other. This causes electronic charge to transfer from the molecular
bond to the hydrogen bond, weakening the covalent molecular
bond and, therefore, decreasing its wavenumber [8]. The second
band is a weak one identified by J (at 1641 cm
1) and assigned as an
out-of-phase stretching motion of C4 = O and C5 = C6 atoms [v
(C4 = O) + v(C5 = C6)]op [10]; this band disappears in the spectrum
recorded at 1.0 GPa. On the other hand, we can observe the increase
of intensity of the band at 1403 cm
1 (marked by L) up to 2.5 GPa.
This band is associated with the scissoring vibration of C20 H2 and
C50 H2 atoms [d(C20 H2)+ d(C50 H2)] [12]. The strongest changes in
the Raman spectra in the region 1350–1730 cm
1 can be observed
between 2.5 and 3.0 GPa. The strong band at 1363 cm
1 (marked by
M) and correlated to the ring stretching vibration coupled with in
plane bending of C6–H atoms [n(ring) + b(C6–H)] [12], splits into
two bands as well as presents abrupt upshift in the spectrum taken
 F.M. Barboza et al. / Vibrational Spectroscopy 89 (2017) 62–68 65

Fig. 4. (a) Raman spectra of thymidine crystals for selected pressures in the spectral region 700–250 cm
1; (b)wavenumber versus pressure plot for the spectral region 700–
250 cm
1.

Fig. 5. (a) Raman spectra of thymidine crystals for selected pressures in the spectral region 250–20 cm
1; (b)wavenumber versus pressure plot for the spectral region 250–
20 cm
1.

at 3.0 GPa (see the bands marked by asterisks). At the same
spectrum we can observe clearly the band at 1358 cm
1 (marked
by M'), which was only a shoulder below 3.0 GPa. Another
important change is that the peak marked by L cannot be seen
in the spectrum recorded at 3.0 GPa. When the pressure is released
back to 1 atm, the spectrum observed (marked with 1 atm* on the
top of Fig. 2(a)) is qualitatively the same as the initial atmospheric
pressure spectrum (bottom of Fig. 2(a)), showing that the pressure-
induced anomalies are not permanent. In this first spectral range is
evident the modifications related to both sugar ring and DNA base
thymine as 3.0 GPa is reached. It will be shown that other spectral
regions also present changes involving thymidine molecule.
The changes in position are illustrated in the wavenumber
versus pressure (v vs P) plots of Fig. 2(b) for the spectral region
1350–1730 cm
1. In particular, Fig. 2(b) shows the discontinuity in
the slope of bands in this spectral region for P  3.0 GPa, with
considerable differences in slopes. It should be emphasized that
dv/dP was found to be positive for most of the modes for
1 atm  P  5.0 GPa, but it is possible to identify negative slope for
the mode marked by the letter I for P < 3.0 GPa and the mode
marked by M' for P > 3.0 GPa. These behaviors are shown in Table 1.
It is worth mentioning that, unless M band all other in this spectral
region have undergone upshift for P  3.0 GPa, even those bands
around 1650 cm
1 (I and J bands), which in the 1 atm < P < 3.0 GPa
66 F.M. Barboza et al. / Vibrational Spectroscopy 89 (2017) 62–68

Table 1
Wavenumber of the peaks observed in the Raman spectra of thymidine for 1 atm, and parameters obtained from linear fitting v = vo + aP to the experimental points as well as
the tentative assignment of the modes.

Peak v[P(1 atm)](cm-1) 1 atm < P < 2.5 GPa 3.0 GPa < P < 5.0 GPa Assignment
Phase I Phase II

v0(cm
1) a(cm
1GPa
1) v[P(3 GPa)](cm
1) a(cm
1 GPa
1)

I 1664 1663.04
5.20 1663 2.89 [n(C4 = O4) + n(C6 = C5)] in phase [10]
J 1641 1641.33 0.40 1651 3.41 [n(C4 = O4) + n(C6 = C5)] outofphase [10]
L 1403 1402.84 2.33 – – d(C20 H2) + d(C50 H2) [12]
M 1363 1363.45 0.2 1358
1.07 n(ring) + b(C6–H) [12]
N' 1233 1233.65 4.57 1242 3.34 n(C5
CH3) + ring[benzene b2u (1308 cm
1)] [10]
N 1225 1224.86 3.78 1222 3.45 n(C5
CH3) + ring[benzene b2u (1308 cm
1)] [10]
O' 1123 1122.58 0.84 – – ring[benzene e1u (1485 cm
1)] [10]
O 1097 1096.37 4.6 – – b(COH) [11]
P 905 905.22 8.2 923 3.25 n(C

C) [11]
Q 898 897.60 0.68 894 2.79 r(C20 H2) [12]
R 852 851.89 3.23 847 2.94 Deoxyribose ring breathing [14]
P' 793 793.50 2.90 797 1.59 ring[benzene b1u (1010 cm
1)] [10]
Q' 772 772.67 2.19 781 1.76 Pyrimidine ring breathing [10]
S' – – – 624
0.05 –
S 543 541.36 4.74 – – –
T 446 445.33 1.83 – – b(COC) [11]
V 421 419.12 0.54 406 4.28 b(COC) [11]
X 396 396.26 7.03 – – g(C5-CH3) [10]
T' 306 305.35 9.92 – – b(C5-CH3) [10]
V' 298 297.83 5.93 296 6.46 b(C5-CH3) [10]
X' 275 274.86 8.38 – – –
H 200 201.28 2.68 219 5.65 Lattice Mode
G 185 184.78 8.84 – – Lattice Mode
F 164 164.45 14.03 – – Lattice Mode
F' 86 85.53 9.15 – – Lattice Mode
* 80 79.90 7.43 – – Lattice Mode
E' 75 74.78 4.56 – – Lattice Mode
E 66 67.19 6.60 – – Lattice Mode
D 59 59.30 6.42 – – Lattice Mode
C 50 49.87 6.95 – – Lattice Mode

b = in-plane bending; g = out-of-plane bending; d = scissoring; r = rocking; n = stretching

pressure range present wavenumber downshift. This should be
expected because these bands are associated with vibrations
where oxygen atoms participate of hydrogen bonds (in this case,
the O(4) oxygen atom). As the crystal reaches the new phase
transfer of charges between molecular bond and HB's have reached
a limit and those covalent bonds become stronger than before the
occurrence of the phase transition (on compression).
Fig. 3(a) presents Raman spectra of thymidine under several
pressure conditions from 1 atm to 5.0 GPa in the spectral range
1300–700 cm
1. On compression we observe that between 1 atm
and 3.0 GPa some modifications are observed. The doublet marked
by N'/N and ascribed as [v(C5
CH3) + ring (benzene b2u)] [10],
starts at about 1233/1225 cm
1 in the 1 atm spectrum and shifts
steadily to about 1242/1222 cm
1 at 3.0 GPa. It is possible to notice
also the inversion of intensity of these bands between 0.5 and
1.0 GPa. By compressing the sample further it is possible to observe
a shoulder centered at 905 cm
1 marked with P, tentatively
assigned as stretching of CC in the furanose [11], which shifts to
higher wavenumbers. Additionally, a clear intensity increase of the
band centered at 1097 cm
1 and marked by the letter O and
associated with a COH bending vibration [11] is observed. In
contrast to the relatively uniform shift of the O mode, the band at
1122 cm
1, marked by O', does not present any unusual change as a
result of compression in the range 1 atm
2.5 GPa, but these two
bands disappear in the spectrum recorded at 3.0 GPa. Furthermore,
a shoulder at 1000 cm
1 (identified by a square) disappears from
the spectrum at 1.0 GPa. The band at 897 cm
1 (marked with Q),
assigned to a rocking of C20 H2 group r(C20 H2) [12], splits into two
bands in the spectrum at 3.0 GPa (marked by asterisks). We can
also observe the splitting of the band at 852 cm
1 (identified by R
and attributed to the ring breathing of deoxyribose [14]) into two
bands (marked by arrows) at 3.0 GPa. Again, in this spectral region
the original spectrum is recovered when the pressure is brought
down to 1 atm.
Fig. 3(b) presents the wavenumber versus pressure plot of the
modes appearing in the 1300–700 cm
1 region of the Raman
spectra of thymidine crystal. We note that many of the observed
Raman modes show minor discontinuities in the wavenumber
slope for the 2.5–3.0 GPa pressure range. Summarizing this
spectral region, it is possible conclude the occurrence of a lot of
chances associated with internal modes as pressure approaches
3.0 GPa. From this one can infer about the deformation of the
thymidine molecules under high pressure conditions.
Fig. 4(a) shows the Raman spectra for several pressure values in
the spectral region 700–250 cm
1 and Fig. 4(b) shows the linear fit
to experimental data. The two bands centered at 276 cm
1 (marked
by X0 ) and at 306 cm
1 (marked by T0 ) decrease their intensities
being not possible to observe them for pressures higher than
2.5 GPa. The bands marked by T' is associated with a bending
vibration, b(C5CH3) vibration [10]. Between 2.5 and 3.0 GPa, it is
possible to observe a linewidth increase and a clear decrease of
intensity of the band assigned with a V0 (at 298 cm
1) and
associated with a b(C5CH3) vibration [10]. On compression we
observe that the band originally at 396 cm
1 (marked by X), which
is associated with an out-of-plane bending vibration, g(C5CH3)
[10], continuously decreases its intensity. In fact, up to pressures at
about 2.0 GPa, it is possible to realize the existence of the two
peaks, X and V (around 420 cm
1), but above 2.0 GPa, certainly,
only a symmetric band appears in the Raman spectrum. Another
important observation deals with the band marked with T (at
446 cm
1) in the spectrum recorded at 1 atm for the pressure range
1 atm
2.5 GPa. Initially it appears as a weak band, but with
 F.M. Barboza et al. / Vibrational Spectroscopy 89 (2017) 62–68
increasing pressure it begins to increase intensity. However, this
band disappears in the spectrum taken at 3.0 GPa. We additionally
note the appearance of two low intense bands marked by S and S0
in the spectrum taken at 0.5 GPa and 3.0 GPa, respectively. When
the pressure is released down to 1 atm (spectrum is marked by
1 atm*) we can observe that the starting original spectrum is
recovered, as can be observed in Fig. 4(a).
In the wavenumber versus pressure plot presented in Fig. 4(b), a
subtle discontinuity occurs in the range 2.5–3.0 GPa, with
appreciable differences in slopes; only one weak mode (marked
by S0 ) presents negative slope. Again, as already noted in other
spectral regions, the spectrum at 3.0 GPa shows that the modes of
thymidine presents strong modifications.
Fig. 5(a) shows the Raman spectra of thymidine crystal
recorded from 1 atm to 5.0 GPa in the 250–20 cm
1 spectral
region, which is pointed in literature as very sensitive to the
symmetry changes of the unit cell. Fig. 5(b) presents the
wavenumber versus pressure plot of the modes appearing in the
250–20 cm
1 region of the Raman spectra of thymidine. Most of
the low-wavenumber modes observed in this region are often
associated with lattice modes. On increasing the pressure, the peak
originally at 59 cm
1 (identified with the letter E) increases
intensity up to 1.0 GPa and then starts to decrease until 2.5 GPa. The
bands marked with C and E present inversion of relative intensity
between 1.0 and 1.5 GPa while the band marked by D decreases
intensity up to 2.5 GPa. However, as 3.0 GPa is reached these bands
seem merge into a single broad band at about 79 cm
1. A similar
behavior occurs with the three bands market by E0 , an * and F0 ;
these bands also merge into a single broad band as shown in the
spectrum recorded at 1.0 GPa. On the other hand, the doublet
centered around 100 cm
1 and 110 cm
1 undergo a splitting
between 0.5 and 1.0 GPa. Another change worth of mentioning is
the apparent merging of the bands F, G and H into only one broad
band at 2.5 GPa. Besides these evidences, the spectrum acquired at
3.0 GPa shows clearly a strong change undergone by the thymidine
crystal, which is correlated with the modifications presented in
other spectral regions. From all those changes is possible to infer
the crystal undergoes a phase transition around 3.0 GPa (from the
atmospheric pressure phase I to phase II). For P > 3.0 GPa the
spectra present low intensity. However, when the pressure is
released back to 1 atm again, the spectrum is qualitatively the same
as that presented in the beginning of the experiment.
From the exposed above, it is possible to conclude that as
3.0 GPa is reached a new unit cell is necessary to maintain the
crystalline structure. However, the results have shown few changes
related to internal modes as well as lattice modes as pressures
around 1.0 GPa is reached. This can be explained as consequence of
small changes associated with the configuration of HBs and little
deformations of the thymidine molecules without any modifica-
tion of the unit cell of the crystal.
Table 1 gives the assignments of the modes (last column) and a
quantitative analysis of the Raman spectra evolution of thymidine
crystal with pressure where fitting of all bands to a linear
expression v = vo + aP is furnished. In Table 1, the second column
(v[P(1 atm)]) represents the wavenumber of the Raman bands
observed at atmospheric pressure, the third and fourth columns,
corresponds to adjustment of data for pressure values between
1 atm and 2.5 GPa and the fifth and sixth columns correspond to
wavenumber fitting in the high-pressure phase, observed between
3.0 and 5.0 GPa.
A study on the DNA bases under high pressure have shown that
the bases adenine, guanine, cytosine and thymine present small
changes related to alterations in the intermolecular HB inter-
actions [15]. The pressure range of the observed changes varies
from 1.6 to 3.1 GPa for the four bases. Compared with thymidine
crystal, the structural modifications of DNA bases are very weak. A
67
possible explanation for this behavior is that, while DNA bases are
constituted of only a ring (cytosine and thymine) or a double ring
(adenine and guanine), the thymidine molecule is constituted of
thymine and sugar ring molecules linked by a covalent bond
involving nitrogen and carbon atoms. This most complex molecule
structure becomes susceptible to suffer conformations under high
pressures.
Pressure-induced comparison between thymidine, adenosine
and cytidine crystals could be useful to clarify about the HBs role in
function of the base. The cytidine crystal presented phase
transition near 4 GPa involving mainly conformations of the ribose
sugar rings. Moreover, changes involving lattice modes were also
observed [8]. On the other hand, adenosine crystal presented a
phase transition around 2.5 GPa involving discontinuities in
internal as well as in lattice modes [7]. For both the cytidine
and the adenosine crystal, the main modifications were observed
with modes associated with the sugar ring. The thymidine crystal,
as shown above, presents a phase transition around 3.0 GPa with
weak HBs rearrangement and conformations of the thymidine
molecules between 0.5 and 1.0 GPa. As analyzed in this study,
modes associated with the sugar ring show the most impressive
anomalies. For instance, three bands increased intensity (O, L and
T) in the range 1 atm
2.5 GPa. However, these same bands
disappear in the spectrum at 3.0 GPa. Two modes (Q and V) have a
discontinuous decrease in their wavenumbers as pressure is
increased through the range 2.5–3.0 GPa. Moreover, it is also
possible to state that the symmetry of the unit cell changes
between phase I to phase II since many modifications are also
observed in the lattice modes region of the Raman spectrum.
The structural stability on compression of the cytidine and
thymine crystals (4.0 GPa and 3.0 GPa, respectively) is greater than
that for the adenosine (2.5 GPa). Obviously, the intensities of
interactions around the nucleosides are important to maintain
their structures under high pressure. Although the number of rings
in the nucleoside molecule is a parameter that should be
considered with further studies involving other nucleosides, a
very important point to be taken into account is the hydrogen bond
network. In the particular case of thymidine, the arrangement of
the hydrogen bonds occurs with the formation of three main bonds
[13]. These hydrogen bonds are formed by the thymine bases with
oxygen atoms of sugars of other molecules; one of the bonds
presents OH  O length of 2.74 Å, where the oxygen atom is
donating the hydrogen. The second hydrogen bond is formed
involving the same oxygen, but now, accepting a hydrogen from a
nitrogen atom in a N

H  O bond with a length of 2.85 Å. The third
hydrogen bond is formed by O

H  O bonds with 2.79 Å with
similar oxygen atoms of different molecules. This set of hydrogen
bonds gives an average length of 2.79 Å. This average length is
small when compared with other organic crystals. For example, the
N

O distance is 2.83 Å for L-alanine, 2.86 Å for L-threonine and
2.90 Å for taurine crystal [16]. A correlation between the lengths of
hydrogen bond and the behavior of the torsion vibration of NH3+
for L-alanine, L-threonine and taurine was obtained in Ref. [16]
showing this bond parameter defines if d n/dP (n corresponds to
the wavenumber) is positive or negative. However, a direct
correlation between the length of hydrogen bond and the pressure
where the crystal undergoes the phase transition was not
established yet. Obviously, it is also possible that beyond the
length, the geometry of the hydrogen bond or even anharmonic
effects could play important roles in the change of the crystalline
structure under different thermodynamics conditions [17]. Al-
though our results do not explore all these aspects, they add
information regarding the behavior of a system dominated by
hydrogen bonds of small dimensions.
Finally, other aspects regarding the phase transition can be
inferred from our results. The first is related to the possibility of
68 F.M. Barboza et al. / Vibrational Spectroscopy 89 (2017) 62–68
coexistence of two phases close to the phase transition pressure.
For example, in the work on adenosine crystal [7], authors have
observed that at 2.63 GPa the presence of two peaks in the Raman
spectrum indicated the simultaneous occurrence of (i) the
atmospheric pressure phase and (ii) the high pressure phase.
Such a fact was also observed with the L-methionine crystal [18],
where it was observed the existence of two different phases at
2.1 GPa. However, in the present study we were not able to find any
indication that a similar coexistence of two phases should be
present on thymidine crystal. The second aspect is related with
eventual cracking of the sample under high pressure conditions.
Previous studies showed that under compression it is possible that
a sample into the diamond anvil cell cracks due the compression
run or even as a result of the decompression process. Such concern
has emerged since the observation that the orthorhombic form of
the L-cysteine crystal cracks into several pieces after the crystal is
being decompressed following a compression run [19]. In the
present case, from a mechanically point of view, the thymidine
crystal remains intact during the whole experiment and we can
state that it remains apparently with no crack. A third aspect –
which has increased in interest in the last years – is related to the
kinetics of application of pressure. It was discovered that with
different rates of compression, it is possible to observe new phases
at high pressure. The most impressive example concerned with L-
alanine that was maintained under pressure for several days at
each pressure value, producing a new phase, while in the situation
when a very rapid compression rate was achieved, the new phase
was not observed [20]. In our study on thymidine crystal we have
maintained the pressure at each point by approximately 1.5 h, what
can not be considered a long time, but enough to induce the
occurrence of one very clear phase transition.
4. Conclusion
Raman spectroscopy was used to study the vibrational
properties of thymidine crystal for pressures up to 5.0 GPa. Little
conformational changes between 0.5 and 1.0 GPa become the
structure of thymidine crystal more stable up to 3.0, as strong
modifications related to both lattice and internal modes indicate
that the material undergoes a phase transition. When the pressure
was released back to atmospheric pressure the Raman spectrum
have shown that the crystal restored its original structure. Finally,
no cracking of the sample was observed in both compression and
decompression runs.
Acknowledgments
Authors acknowledge CNPq for financial support [process
490343/2013-9] and FUNCAP/PRONEX.
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