Physical Chemistry Chemical Physics
Page 6 of 12
ARTICLE
DOI: 10.1039/C5CP03615A
absorption coefficients26. The generation, as a minor product, of a
covalent SꢀS bridged dimer formed by two S4TdR molecules could
be responsible for those spectral features. It is worth noting that
thisspecies has been already observed after long term exposure of
S4TdR to the same light source adopted during the present
investigation but in the absence of RB8.
medium is 2.4×10ꢀ4 M, and the constant of RB triplet quenching by
S4TdR estimated above is < 108 Mꢀ1 sꢀ1, it can be safely concluded
that almost 100% of RB triplet is quenched by molecular oxygen
under aerobic conditions,even at the highest concentration of S4TdR
used. Therefore, the luminescence quenching observed in Figure 3
can be reasonably attributed to a chemical process, responsible for
the formation of S4TdR oxidation products.
FTIRꢀATR data qualitatively similar to those described so far were
obtained for the S4TdR/RB2×10ꢀ4/5×10ꢀ4 M reaction mixtures at pH
7 and 12 (data not shown).
FTIR-ATR spectroscopy measurements
FTIRꢀATR, 1HꢀNMR and ESIꢀMS measurements were performed on
irradiated S4TdR/RB mixtures in order to confirm the presence of
thymidine, clearly indicated by UVꢀVis data, and to detect
eventualfurther compounds arising from S4TdR photodegradation
but not exhibiting a peculiar absorption band in UVꢀVis spectra. In a
preliminary step of the FTIRꢀATR study, spectra were acquired on
the RB 5 × 10ꢀ4 M solution at pH 7 and 12 and are reported in
Figures S3a and S3d of the Supporting Information, respectively.
1H-NMR spectroscopy measurements
The 1HꢀNMR spectrum obtained before irradiation (0 h) for the
S4TdR/RB 8×10ꢀ4/5×10ꢀ4 M aqueous solution at pH 7 is reported in
Figure 4. As already explained, a considerable reduction of the
intense signal due to the water protons(4.77 ppm) was achieved
during this study by using the pulse sequence reported in the
experimental section, thus avoiding the need for a deuterated
solvent. The main signals related to S4TdR (each marked by an S
followed by the indication of the corresponding hydrogen, numbered
as in the S4TdR structure reported in Figure 4), were assigned
according to Zhang et al27and have been recently discussed in detail
in Ref. 8. The further signal detected in the spectrum, labeled simply
as R, was attributed to the RB molecule28,using, as a reference, the
spectrum acquired, under the same instrumental conditions, for a RB
5×10ꢀ4 M solution at pH 7 (data not shown).
In both cases the comparison between spectra recorded before (time
0) and after 1 hour of irradiation with the neon lamp adopted during
this studyis shown. Bands at 1340, 1450 and 1545 cmꢀ1, formerly
ascribed to C=C stretching25, and the one at 1600 cmꢀ1, likely due to
C=O groups, observed at pH 7 / time 0, are characteristic of
xanthene dyes. A remarkable change of the spectral features
occurred at pH 12, where the 1450 cmꢀ1 band became larger, shifted
to 1425 cmꢀ1 and significantly more intense than the others. This
effect could be due to the more extended conjugation resulting from
the basic pH. Not surprisingly, a general increase of transmittance
occurred after irradiating the RB solution for 1 hour (dark grey lines
in Figures S3a and S3d), suggesting that a slight RB bleaching took
place, as also observed in the UVꢀVis spectra (Figure 1b), likely
accompanied by variations in electron delocalization in the case of
pH 12.
FTIRꢀATR spectra observed for the S4TdR/RB reaction mixtures, as
those reported in Figures S3b and S3e, were quite complex, due to
the superposition with S4TdR spectral features, recently described
for the same system in the absence of RB8. The main S4TdRꢀrelated
bands, located at 1625 and 1690 cmꢀ1 and ascribed to C2=O and
C5=C6 stretching, respectively8, were clearly visible for the nonꢀ
irradiated solution at pH 7 (black line in Figure S3b). When the nonꢀ
irradiated S4TdR/RB mixture at pH 12 was considered no significant
modifications were present with respect to the RB spectrum (black
line in Figure S3e), since the main absorption band of S4TdR at that
pH was located exactly at the same wavenumber (1425 cmꢀ1) as the
main RB band8. After 1 hour of irradiation of the mixture containing
8×10ꢀ4 M S4TdR and 5×10ꢀ4 M RB at pH 7 the valley between the
characteristic bands of S4TdR appeared much less evident (grey line
in Figure S3b), suggesting the presence of a contribution due to the
main band of TdR (1690 cmꢀ1 at pH 7, see Figure S3c). At pH 12,
after 1 hour of irradiation (grey spectrum in Figure S3e), the increase
of absorption at 1600 and 1650 cmꢀ1, i.e. the wavenumbers related to
the TdR bands not superimposed to those due to RB or residual
S4TdR at that pH (see Figure S3f), suggested that thymidine had
been generated from S4TdR. Consequently, FTIRꢀATR data
confirmed TdR to be the main product of S4TdRphotoxidation both
at pH 7 and pH 12. However, some minor variations occurring in the
FTIRꢀATR spectra after 1 hour of irradiation of the S4TdRꢀRB
solutions suggested the presence of a further reaction product. In
As shown in Figure 4, several new signals appeared in the spectrum
after 1 hour of irradiation (1 h). Some of them, labeled with a T,were
easily related to TdR (see the numbering of hydrogen atoms in the
TdR structure reported in the bottom part of Figure 4) through a
comparison witha spectrum obtained, under the same experimental
conditions, for a thymidine solution containing RB (data not shown).
Not surprisingly, most of the signals related to the deoxyribose ring
ofS4TdR and TdR were very close, even superimposed; indeed, the
replacement of an oxygen atom with a sulphur one on the nucleoside
basecan influence the chemical shift of the deoxyribose protons
onlyslightly. Interestingly, two additional signals (indicated by
dotted arrows in Figure 4) were observed, close to S4TdR ones, in
the spectrum obtained after 1 h of irradiation at pH 7 and have been
emphasized in the two spectral magnifications reported in Figure
4.Their correlation with a further compound having a thiothymidineꢀ
like (SꢀLike) structure could be hypothesized, based on their
chemical shifts, 8.085 ppm and 2.060 ppm, respectively. Indeed,
1
signalsclose to these shifts were detected in the HꢀNMR spectrum
of S4TdR before irradiation and assigned to its6ꢀH and 7ꢀCH3
protons, respectively (see the 0 h spectrum in Figure 4). Moreover,
the ratio of the integral areas related tothe two new signalswas in
excellent agreement with the 1:3 proportion predicted between the 6ꢀ
H and the 7ꢀCH3 protons of S4TdR. It is also worth noting that the
chemical shift variation(from 7.77 to 8.085 ppm)observed forthe
new 6ꢀH proton suggested the presence of a structure with an
enhanced aromatic character, in which carbon 6 perceived a higher
deshielding effect with respect to its counterpart in S4TdR. The
presence of a S4TdR dimer, already suggested by FTIRꢀATR data,
would be compatible with this finding. It is also worth noting that
the weak additional signals observed in the 1 h spectrum, with
chemical shifts close to those related to some of the ribose ring
protons of S4TdR and TdR (i.e., 3’ꢀH, 4’ꢀH and 5’ꢀH), are likely
related to the ribose rings protons of the dimeric species.
particular,
a slight transmittance decrease was observed at
wavenumbers lower than 1000 cmꢀ1, especially at pH 7 (see the grey
spectrum in Figures S3b and S3e). Interestingly, absorptions below
1000 cmꢀ1 have been often reported for the CꢀS and SꢀS stretching,
although their detection is generally difficult, due to the low molar
The 1HꢀNMR spectrum obtained for the pH 7 reaction mixture
initially containing 2×10ꢀ4 M S4TdR and 5×10ꢀ4 M RB, before and
after 1 hour of irradiation, showed spectral features similar to those
6 | J. Name., 2012, 00, 1-3
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