Xanthines studied via femtosecond fluorescence spectroscopy

Article abstract of DOI:10.3390/molecules21121668

Xanthines represent a wide class of compounds closely related to the DNA bases adenine and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical purposes was proposed, their fluorescence properties have not been properly characterized so far. The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits non-exponential fluorescence decays with no significant dependence on the emission wavelength. The fluorescence quantum yield (3 × 10^-4) and average decay time (0.9 ps) are slightly larger than those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence anisotropies vary only weakly with the site and the degree of methylation. These findings are in line with theoretical predictions suggesting the involvement of several conical intersections in the relaxation of the lowest singlet excited state.

Full text of DOI:10.3390/molecules21121668

molecules
Article
Xanthines Studied via Femtosecond
Fluorescence Spectroscopy
Pascale Changenet-Barret 1,†, Lajos Kovács 2,*, Dimitra Markovitsi 1 and Thomas Gustavsson 1,
*
1
LIDYL, CEA, CNRS, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France;
pascale.changenet-barret@polytechnique.edu (P.C.-B.); dimitra.markovitsi@cea.fr (D.M.)
Department of Medicinal Chemistry, University of Szeged, Dóm tér 8, 6720 Szeged, Hungary
2
*
Correspondence: kovacs.lajos@med.u-szeged.hu (L.K.); thomas.gustavsson@cea.fr (T.G.);
Tel.: +36-62-545145 (L.K.); +33-1-6908-9309 (T.G.); Fax: +36-62-545971 (L.K.); +33-1-6908-8707 (T.G.)
Present address: LOB, Ecole Polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128
Palaiseau, France.
†
Academic Editor: Carlos E. Crespo-Hernández
Received: 14 October 2016; Accepted: 29 November 2016; Published: 3 December 2016
Abstract: Xanthines represent a wide class of compounds closely related to the DNA bases adenine
and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double
helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical
purposes was proposed, their fluorescence properties have not been properly characterized so far.
The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond
spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits
non-exponential fluorescence decays with no significant dependence on the emission wavelength.
The fluorescence quantum yield (3
×
10−4) and average decay time (0.9 ps) are slightly larger than
those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties
of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence
anisotropies vary only weakly with the site and the degree of methylation. These findings are in
line with theoretical predictions suggesting the involvement of several conical intersections in the
relaxation of the lowest singlet excited state.
Keywords: xanthines; femtosecond spectroscopy; fluorescence; electronic excited states
1. Introduction
The term “xanthines” denotes a wide class of compounds whose central core (3,7-dihydro-
purine-2,6-dione) is closely related to the DNA bases guanine and adenine (Figure 1). Like nucleobases,
xanthines are capable of forming hydrogen bonds, allowing their insertion in duplexes [
quadruplexes (G-quadruplexes) [ ]. Moreover, it has been shown that their self-association can give
rise to four-stranded structures [ ], which have attracted attention for applications in the field
of molecular electronics [ ]. Xanthines are ubiquitous in the human body, where they are formed
following enzymatic degradation of adenine and guanine. Moreover, methylxanthines are used as
therapeutic agents acting, among others, as stimulants of the nervous system [10 11]. The well-known
1] and guanine
2–4
2,5–8
9
,
caffeine, present in coffee and tea and various soft beverages, is none other than 1,3,7-trimethylxanthine
(Figure 1). It is also worth noticing that 20-deoxyxanthosine (dX) has been used in the extension of the
genetic alphabet by purine pairing with a 2,4-diaminopyrimidine nucleoside, which has a hydrogen
bonding pattern complementary to dX [12].
Despite the omnipresence of xanthines in our life, very few studies discuss their intrinsic
fluorescence properties. Shukla and Mikla examined the effect of pH on the fluorescence spectra
of several xanthines [13]. McKemy et al. reported that the determination of calcium release in cells by
Molecules 2016, 21, 1668; doi:10.3390/molecules21121668
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measuring the fluorescence signal of the dye indo-1 may be perturbed by the intrinsic fluorescence of
caffeine [14]. Karim et al. developed an array for the simultaneous determination of acetylsalicylic
acid and caffeine in commercial tablets exploiting their natural fluorescence [15]. Wei et al. studied
the fluorescence performance of three methylxanthine derivatives at low and room temperatures and
reported fluorescence quantum yields in fluid solutions ranging from 0.2 to 0.6 [16]. Surprisingly, these
values are higher by at least three orders of magnitude than those determined for the DNA bases in
solution [17]. So far, no xanthine fluorescence lifetime is available in the literature. Therefore, in view
of the possible analytical applications, and because of the potential use of xanthines as artificial letters
of the genetic code and building blocks of G-quadruplexes, a better understanding of their fluorescence
dynamics, and thereby the electronic state relaxation processes, is necessary.
The objective of the present study is to provide a first insight into the dynamical properties
of xanthine fluorescence. The main investigation tool is femtosecond fluorescence spectroscopy
using fluorescence upconversion, a technique widely proved valuable for the study of DNA bases
whose fluorescence is extremely short-lived [17,18]. We examine seven methylated xanthines
characterized by different methylation sites and various degrees of methylation (Figure 1), four
monomethylated derivatives: 1-methylxanthine (1-MX), 3-methylxanthine (3-MX), 7-methylxanthine
(7-MX), and 9-methylxanthine (9-MX); two dimethylated ones: 1,3-dimethylxanthine (1,3-DMX,
theophylline) and 3,7-dimethylxanthine (3,7-DMX, theobromine), and one trimethylated compound:
1,3,7-trimethylxanthine (1,3,7-TMX, caffeine).
Figure 1. Schematic representation of the studied methylxanthines and of the DNA nucleosides dA and
dG. Canonical numbering for the purine ring is noted in the structural formula of 1-methylxanthine,
0
while that of the sugar ring is shown in the structural formula of 2 -deoxyadenosine (dA).
In the first part, we focus on 3-MX, providing fluorescence spectra and quantum yields in
methanol and water as well as fluorescence decays. We compare its behavior with those of the
adenine and guanine 20-deoxynucleosides, dA and dG. Our comparison is made with nucleosides
instead of nucleobases because, on the one hand, the solubility of guanine is too low for time-resolved

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measurements [19] and, on the other hand, the presence of two tautomers renders the study of
adenine in solution difficult [20]. Subsequently, we present fluorescence decays and fluorescence
anisotropy decays obtained for all the examined compounds in methanol. We chose this solvent for
our comparative time-resolved fluorescence measurements because some of the studied xanthines
are barely soluble in water, giving rise to aggregates. Finally, we discuss their excited state
relaxation in association with the scarce studies tackling this topic, performed by quantum chemistry
methods [21,22] and transient absorption spectroscopy [23,24].
2. Results
2.1. Fluorescence Properties of 3-Methylxanthine
Figure 2 shows the steady-state absorption and fluorescence spectra of 3-MX in water and
methanol. The absorption spectra in these two solvents are similar, both peaking at 271 nm.
The fluorescence spectrum of 3-MX in methanol, which shows a maximum at 311 nm, is slightly
blue-shifted from that in aqueous solution, peaking at 315 nm. The spectral halfwidth ∆ν, determined
after conversion of the fluorescence spectra to a wavenumber scale (multiplying by a factor λ
2), is
significantly narrower for 3-MX in methanol (5200 cm−1) compared to water (6150 cm−1). All these
spectral features are gathered in Table 1, together with those of dA and dG in water reported
previously [17,25]. The fluorescence quantum yield of 3-MX in water is more than double those
of dA and dG determined under the same conditions, but the spectral width is twice as narrow. Note
also that 3-MX was found to be about twice as fluorescent in methanol as in water.
Figure 2. Normalized steady-state absorption and fluorescence spectra of 3-MX in methanol (black)
and water (red) on a wavelength scale normalized to their respective maxima. Excitation wavelength:
267 nm (indicated by a vertical dashed line).
Table 1. Comparison of the steady-state fluorescence properties of 3-MX in methanol with those
of dA and dG in water: fluorescence maximum (λfl), fluorescence quantum yield (φf), and spectral
width (∆ν).
−1
Compound/Solvent
λfl (nm)
φf × 104
∆ν (cm
)
3-MX/MeOH
3-MX/H2O
dA/H2O
311
315
307 1
334 1
1
4.5
2.4
5200
6150
10,150
11,390
0.9 1
1.0 1
dG/H2O
Onidas et al. [17].

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Figure 3a displays the total fluorescence decays of 3-MX measured at four wavelengths along
with those of dA and dG measured at 330 nm, close to their emission maxima. Also shown in this
figure is the 330 fs (fwhm) instrument response function. The fluorescence decays of the xanthine
are significantly slower than those of the nucleosides. The dependence of the fluorescence dynamics
of 3-MX with the emission wavelength is negligible, except at 310 nm where a slightly faster decay
is observed.
The fluorescence anisotropies of 3-MX and the two nucleosides at 330 nm are shown in Figure 3b.
The 3-MX anisotropy is somewhat higher than that of dA, while a larger difference is observed with
respect to that of dG.
a
b
Figure 3. (a) Normalized total fluorescence decays recorded at 310 nm (blue), 330 nm (red), 350 nm
(violet), and 370 nm (green) for 3-MX in methanol, compared to those recorded for dA (grey; 330 nm)
and dG (black; 330 nm) in the same solvent. The instrument response function is displayed as a dashed
line. Excitation wavelength: 267 nm. (b) Fluorescence anisotropy decays at 330 nm of 3-MX (blue), dA
(red), and dG (green) in methanol. Solid lines, derived from fits with mono-exponential functions, are
only shown to guide the reader.
As found for the DNA nucleosides and nucleotides [17
be fitted by mono-exponential functions. Bi-exponential functions exp(
provided satisfactory fits. The τ1 and τ2 values are, respectively, in the 0.7–1.11 ps and 2.8–5.5 ps
ranges. In Table 2, we compare the average fluorescence lifetimes: < τ2) of 3-MX
> = ατ1 + (1 − α
with those of dA and dG derived from the fits of the decays at 330 nm. It appears that the < > value of
,
19], the fluorescence decays of 3-MX cannot
−
t/τ1) + (1 − α)exp( t/τ2
−
)
τ
)
τ
3-MX in methanol is higher than those of dA (by a factor of 6) and dG (by a factor of 4) in the same
solvent. For both nucleotides, when going from methanol to water, the average lifetime decreases. It is
also interesting to consider the radiative lifetime τrad, obtained by dividing the fluorescence lifetime by
the fluorescence quantum yield [26]. The radiative lifetime is an intrinsic property of the chromophore,
directly associated with the oscillator strength, and thereby the electronic structure. This allows a
comparison of the different compounds in different environments. Interestingly, the radiative lifetime
of 3-MX (3.7 ns) is quite close to that of dG (3.3 ns) but twice as long as that of dA (1.4 ns). All these
values correspond, however, to highly allowed transitions.
Table 2. Comparison of the fluorescence lifetimes of 3-MX in methanol at 350 nm with those of dA and
dG in the same solvent at 330 nm: average fluorescence lifetime <τ> and radiative lifetime (τrad).
Compound/Solvent
<τ> (ps) 1
τrad (ns)
3-MX/MeOH
dA/H2O
dA/MeOH
dG/H2O
1.67
3.7
1.4
nd
3.3
nd
0.13 2
0.27 3
0.33 4
0.42 3
dG/MeOH
1
<τ> = ατ1 + (1 − α)τ2; 2 Onidas et al. [17]; 3 present work; 4 Miannay et al. [19].

Molecules 2016, 21, 1668
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2.2. Substituent Effect on the Fluorescence Dynamics
The effect of monomethylation on the fluorescence decays of the xanthine is illustrated in Figure 4a
while that of di- and trimethylation is shown in Figure 5. In all cases, good fits required bi-exponential
model functions. This is illustrated for 1-MX in Figure 4b. The parameters derived from these fits are
given in Table 3.
a
b
Figure 4.
in methanol after excitation at 267 nm: 1-MX (blue), 3-MX ((violet), 7-MX (red) and 9-MX (green).
) Parallel and perpendicular fluorescence decays of 1-MX in methanol recorded at 310 nm after
(a) Normalized fluorescence decays recorded at 350 nm for monomethylated xanthines
(
b
excitation at 267 nm. Also shown are the fitted curves using mono-exponential (dashed line) and
bi-exponential (solid line) model functions.
Figure 5. Effect of the degree of methylation on the (normalized) fluorescence decays of xanthines
recorded at 350 nm: 3-MX (violet), 1,3-DMX (blue), 3,7-DMX (green), and 1,3,7-TMX (red).
Table 3. Parameters derived from the fits of the fluorescence decays and anisotropy decays measured
at 350 nm after excitation at 267 nm for methylated xanthines in methanol.
Compound
α
τ (ps)
1
τ (ps)
2
<τ> (ps) 1
r0
τTA (ps) 2
1-MX
3-MX
7-MX
0.82
0.49
0.49
0.68
0.51
0.35
0.65
0.54
0.66
0.44
0.52
0.60
0.71
0.85
2.62
2.65
1.72
2.50
1.69
2.70
2.35
2
0.91
1.67
1.10
1.15
1.13
2.01
1.37
0.34
0.33
0.32
0.31
0.33
0.32
0.33
9-MX
1,3-DMX
3,7-DMX
1,3,7-TMX
1.7
2.0
1.3
1
average fluorescence lifetime <
transient absorption [23].
τ
> = τ1 + (1 − α
)τ2
;
monoexponential decay time observed in acetonitrile by

Molecules 2016, 21, 1668
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It appears that, while methylation at position 1 results in the shortest decay (<
τ> = 0.91 ps),
methylation at position 3 increases the lifetime by a factor of ~2 (<τ> = 1.67 ps). 7-MX and 9-MX exhibit
intermediate lifetimes of 1.10 ps and 1.15 ps, respectively.
Going from monomethylated to dimethylated derivatives, we observe that the <
τ> value obtained
for 1,3-DMX (1.13 ps) corresponds roughly (within 15%) to the average of the < > values (1.29 ps) found
τ
for 1-MX (0.91 ps) and 3-MX (1.67 ps). In contrast, the lifetime of 3,7-DMX (2.01 ps) is significantly
longer than the average value (1.45 ps) found for the corresponding monomethylated xanthines (1.67 ps
for 3-MX and 1.10 ps for 7-MX). Surprisingly, the addition of a third methyl group at position 1 shortens
the fluorescence lifetime (<τ> = 1.37 ps for 1,3,7-TMX).
The average fluorescence lifetimes obtained for the di- and trimethylated xanthine derivatives are
close to the lifetimes reported by Chen and Kohler [23], also shown in Table 3. The latter were derived
from mono-exponential fits of the transient absorption signals obtained for acetonitrile solutions.
The effect of the xanthine monomethylation on the fluorescence anisotropy is shown in Figure 6a
while the anisotropy signals of di- and trimethylated xanthines are shown in Figure 6b. As illustrated
by the figure, the anisotropies of the four compounds are similar. Comparable anisotropy decays were
also observed for the di- and tri-methylated xanthines. The merged nonlinear fitting/deconvolution
process of the measured (Ipar) and (Iperp) signals, as described in the Materials and Methods section,
allowed us to extract the time-zero anisotropies r0, shown in Table 3. The r0 values found for all
studied xanthines are around 0.33. Note that, due to the very short fluorescence decays of the studied
compounds (<3 ps), it was not possible to extract the rotational diffusion times, which are expected to
be about 20–30 ps.
Figure 6. Fluorescence anisotropy decays determined at 350 nm after excitation at 267 nm for (
a) the
monomethylated xanthines: 1-MX (blue), 3-MX (violet), 7-MX (red) and 9-MX (green) and for (
and tri-methylated xanthines: 1,3-DMX (blue), 3,7-DMX (red) and 1,3,7-TMX (blue).
b) di-
3. Discussion
In the first part of our study, we found that the 3-MX absorption spectrum is characterized by
a strong band peaking at 271 nm. This corresponds to the first ππ* transition. Quantum chemistry
calculations on various methylated xanthines have shown that the next ππ* transition is situated about
1 eV higher [13,22]. Thus, excitation at 267 nm populates only the lowest excited ππ* state of 3-MX.
This contrasts with dG, and to a lesser extent dA, for which two near-lying ππ* states are populated
at 267 nm. Subsequently, internal conversion between these two ππ* states leads to depolarization
faster than our time-resolution (100 fs after deconvolution). Consequently, the time-zero anisotropy
determined for the nucleosides (0.15 for dG and 0.24 for dA, see ref. [17]) is lower than the 0.4 value
corresponding to parallel absorption and emission dipoles. Yet, the r0 value determined for 3-MX,
as well as for all the studied xanthine derivatives (0.31–0.34), is still lower than the theoretical limit.

Molecules 2016, 21, 1668
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Such a discrepancy may be explained by vibrational relaxation in the excited state. A similar effect
was noticed for the extensively studied thymine chromophore [27].
The fluorescence lifetime of 3-MX in methanol was found to be 1.67 ps, while the radiative lifetime
is estimated to about 3.7 ns (1.67 ps/4.5
×
10−4, from Tables 1 and 2). These findings allow us to
draw two important conclusions: (i) the emission corresponds to a strongly allowed ππ* electronic
transition, as for the DNA mono-nucleosides and mono-nucleotides [17]; (ii) the 3-MX excited state is
very efficiently depopulated by non-radiative processes.
Our results are in line with those reported recently from transient absorption measurements [23],
showing that all studied methylxanthines undergo ultrafast excited state relaxation. Despite their
slightly slower fluorescence decay, their behavior is very similar to that of the DNA/RNA building
blocks [18,28]. In the introduction, we evoked the structural similarities between xanthine and
the purines adenine/guanine. In Table 2, it is evident that the average florescence decay of 3-MX
(a “typical” mono-methylated xanthine) is about five times slower than those of dA and dG. This hints
at a possible difference in deactivation mechanism between the xanthine and the two natural DNA
building blocks. At the same time, the 1.67 ps time constant of 3-MX corresponds to an “ultrafast”
excited state deactivation, which means that the presence of a conical intersection is highly probable.
Numerous theoretical studies have attributed this ultrafast excited state relaxation to very efficient
non-radiative processes involving easily accessible conical intersections (CIs) connecting the first ππ
*
excited state with the ground state. For a recent review on theoretical studies of the excited states
of nucleobases, see [29]. These processes are accompanied by an out-of-plane motion of peripheral
substituents, at the 2-position for purines and the 5-position for pyrimidines [18]. A systematic study
of the substituent effect had been performed via femtosecond fluorescence spectroscopy for uracil
derivatives [30]. Only methylation at the 5-position was found to significantly increase the excited
state lifetime (by a factor of 4) of uracil (0.39 ps vs. 0.10 ps) in line with an out-of-plane motion of the
C5 carbon [30–34].
As can be seen in Figure 1, the 2-position in xanthines is blocked by a carbonyl group and the
5-position by the imidazole ring. Yet, the average lifetimes reported in Table 3 for monomethylated
xanthines show a weak dependence on the methylation site. While the <τ> values of 1-MX, 7-MX, and
9-MX are close to 1 ps (Table 3), that of 3-MX jumps up to 1.7 ps, suggesting that the substitution at
the 3-position significantly affects the excited state dynamics. In this regard, it should be recalled that
3-substituted xanthines are potentially tetrad- and G-quadruplex-forming compounds [2].
The effect of the substitution in the 3-position is, however, far from being trivial in di- and
trimethylated xanthines. The longest lifetime of 2.0 ps is observed for 3,7-DMX (theobromine), while
1,3-DMX (theophylline) and 1,3,7-TMX (caffeine) exhibit faster decays than that of 3-MX. We stress
that, for the monomethylated compounds, methylation at the 1- or the 7-position has very little
effect (<20%) on the excited state lifetime. This can be taken as a strong indication that the excited
state deactivation of xanthines cannot be reduced to the involvement of one single localized bond.
Interestingly, Nachtigallova et al. considered the existence of two different conical intersections in the
excited state deactivation of theophylline, theobromine, and caffeine: (i) an out-of-plane deformation
of the five-membered imidazole ring, and (ii) an out-of-plane deformation of O6 on the six-membered
ring [22]. In addition, direct or indirect Franck-Condon-CI reaction paths were found to be energetically
allowed, leading to potentially very complex excited state dynamics. In this context, the peculiar
influence of methylation observed above is not surprising. The above calculations predicted reaction
paths involving different energy barriers. The smallest barrier was found for 1,3-DMX (0.2 eV),
followed by 1,3,7-TMX (0.6 eV) and 3,7-DMX (1.2 eV). This energetic ordering is in excellent agreement
with the observed fluorescence lifetimes for these three compounds, 1.13 ps, 1.37 ps, and 2.01 ps,
respectively, even though the height of these barriers is not necessarily compatible with picosecond
lifetimes. It should also be kept in mind that solvent effects, which were not considered in these
calculations, can lower the energy barriers [31].

Molecules 2016, 21, 1668
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As already pointed by Chen and Kohler [23], the xanthine structure is closely related to that of
uracils. Both molecules contain a heteroatomic 6-membered ring with two carbonyl oxygens in the same
positions (positions 2 and 6 for xanthines, positions 2 and 4 for uracils) and one double bond (C4-C5
for xanthine, C5-C6 for uracil). The major difference is the presence of a five-membered imidazole ring
attached to the C4-C5 double bond in xanthines. Considering the strong involvement of the C5-C6
double bond in the non-radiative deactivation mechanism of uracils, the little influence of the imidazole
ring on the “uracil-like” structure of xanthines is surprising. The excited state dynamics of uracils
leading to the conical intersection have often been described as an ethylene-like torsional movement,
bringing both the 5- and the 6-substituents out-of-plane in an opposite manner [35]. The “blocking”
effect of substitution on the 5-position is today well understood, and explained by the out-of-plane
motion of the substituent [30]. Blocking the C5-C6 double bond inhibits efficiently the non-radiative
decay of uracils as shown by Lim and coworkers in the case of 5,6-trimethyleneuracil (TMU), for
which the excited state lifetime increases to several tens of picoseconds [36]. The non-radiative decay
mechanism taking place in the structurally very close molecule cytosine involves a similar C5-C6
activity; by constraining the double bond in 5,6-trimethylenecytosine (TMC), the excited state lifetime
increases dramatically from 0.3 ps to 1.2 ns [36]. Such a “blocking” effect is not observed in the case
of the investigated methylxanthines. Its absence can be explained by the internal dynamics of the
imidazole ring that is largely responsible for the non-radiative decay [22].
From the above discussion, it appears that it is difficult to generalize mechanistic schemes
between closely related purines and pyrimidines. The decay paths of each heteroatomic system
must be treated individually. The fingerprint of the decay path, depending on the topology of the
Potential Energy Surface (PES) of the emitting state, is actually reflected in the width of the steady-state
fluorescence spectra (Table 1). For both dG [37] and dA [25], quantum chemistry calculations predict
a pronounced “flat” and barrierless shape of the PES. This induces a substantial spreading of the
wavepacket during its motion towards the conical intersection resulting in a spectral broadening of
the fluorescence. For 3-MX, on the other hand, the substantially longer fluorescence lifetime is in line
with the presence of a barrier on the PES, confining the excited state population and giving rise to a
narrower emission spectrum.
4. Materials and Methods
1-Methylxanthine (1-MX) and 9-methylxanthine (9-MX) have been obtained from Molekula
GmbH (Garching bei München, Germany), 3-methylxantine (3-MX) from TCI Europe N.V.
(Zwijndrecht, Belgium), theophylline (1,3-DMX), theobromine (3,7-DMX), and caffeine (1,3,7-TMX)
from Sigma-Aldrich (Budapest, Hungary). 7-Methylxanthine (7-MX) was synthesized by modifying
the procedure by Bridson et al. [38]. Thus, guanosine was methylated with methyl iodide and
then hydrolyzed with hydrochloric acid. The resulting 7-methylguanine hydrochloride was treated
◦
with sodium nitrite in acetic acid, and the product was recrystallized from water (mp. > 360 C).
The 1H-NMR spectrum of 7-MX obtained in this way was identical to that described by Müller et al. [39].
Steady-state absorption and fluorescence spectra were recorded with a Perkin-Elmer Lambda
900 spectrophotometer (Bodenseewerk, Perkin-Elmer Gmbh, Überlingen, Germany) and a SPEX
Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon Inc., Edison, New Jersay, USA). Methanol for
UV spectroscopy was obtained from Fluka (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
Fluorescence quantum y−ie4lds were determined using TMP (thymidine monophosphate) in water as a
reference (
Φ
F = 1.54
×
10
, λexc = 255 nm) [17]. TMP was obtained from Sigma-Aldrich (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) and ultrapure water delivered by a Millipore Milli-Q Synthesis
Millipore SAS, Molsheim, France) system.
Time-resolved fluorescence measurements were carried out with the fluorescence up-conversion
technique (FU) using the third harmonic (267 nm) of a Ti/sapphire laser system (Coherent MIRA,
76 MHz, 120 fs, 800 nm) for the excitation [27].

Molecules 2016, 21, 1668
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The average excitation power was 60 mW. The sample fluorescence was collected with two
parabolic mirrors and mixed with the residual 800 nm fundamental beam in a 0.5 mm type I BBO crystal.
The up-converted signal was then dispersed in a double-grating monochromator (spectral resolution
ca. 5 nm) and detected by a photomultiplier in single photon counting mode. The fluorescence decays
were recorded in a 10 ps time window, with parallel and perpendicular excitation-detection polarization
configurations by adjusting the excitation beam polarization with a zero-order half-wave plate.
The total fluorescence kinetics F(t) shown in Figures 3–5 were constructed from the parallel, Ipar(t),
and perpendicular, Iperp(t), signals according to the equation F (t) = Ipar (t) +
2
I
prep (t). Likewise, the
ꢀ
ꢁ
fluorescence anisotropy r(t) is given by the expression: r (t) = Ipar (t) − Iprep (t) /F (t).
In order to evaluate the characteristic times involved, instead of treating F(t) and r(t) separately,
we performed a merged nonlinear fitting/deconvolution process using the impulse response model
functions
instrument response function, I(t)
to the experimentally measured Ipar(t) and Iperp(t) signals. The full width at half maximum (fwhm) of
i
par (t) = (1 + 2r (t)) f (t) and
i(t)
i
⊗
prep (t) = (1 − r (t)) f (t), convoluted by the Gaussian
∝
G(t). The model functions thus obtained were fitted
the instrument response function was found to be about 350 fs fwhm at 330 nm.
5. Conclusions
Our study shows that, in terms of fluorescence, methylated xanthines behave very much as
DNA nucleosides and nucleosides. Their room temperature solutions are very weakly emitting, and
their fluorescence decays, being extremely short lived, can be determined only using femtosecond
techniques. The position and the degree of methyl substitution do not influence dramatically the
excited state deactivation rate. However, the fluorescence properties reported in the present work
concern xanthines as monomeric chromophores. In environments where their solubility is low, or
at high concentrations, self-aggregation may occur, leading to important changes in the nature of
the excited states and, consequently, on the fluorescence properties. Indeed, it has been shown that
guanine quadruplexes, due to strong electronic interactions among the bases giving rise to collective
excited states, exhibit higher fluorescence quantum yields and longer fluorescence lifetimes than
isolated guanosines in solutions [40–42]. In order to evaluate such effects in the case of xanthines,
specific studies focusing on self-associated structures are needed. This is a prerequisite in order to use
their fluorescence for analytical purposes.
Acknowledgments: This study was supported by a short mission grant in the frame of the COST Action MP0802
(Self-assembled guanosine structures for molecular electronic devices).
Author Contributions: P.C.-B, L.K., D.M., and T.G. conceived and designed the experiments; L.K. synthesized
7-MX; P.C.-B., T.G., and L.K. performed the experiments; P.C.-B. and T.G. analyzed the data; D.M. and T.G. wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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