Converging fate of the oxidation and reduction of 8-thioguanosine

Article abstract of DOI:10.3390/molecules24173143

Thione-containing nucleobases have attracted the attention of the scientific community for their application in oncology, virology, and transplantology. The detailed understanding of the reactivity of the purine derivative 8-thioguanosine (8-TG) with reactive oxygen species (ROS) and free radicals is crucial for its biological relevance. An extensive investigation on the fate of 8-TG under both reductive and oxidative conditions is here reported, and it was tested by employing steady-state photooxidation, laser flash photolysis, as well as γ-radiolysis in aqueous solutions. The characterization of the 8-TG T₁ excited state by laser flash photolysis and the photooxidation experiments confirmed that singlet oxygen is a crucial intermediate in the formation of the unexpected reduced product guanosine, without the formation of the usual oxygenated sulfinic or sulfonic acids. Furthermore, a thorough screening of different radiolytic conditions upon γ-radiation afforded the reduced product. These results were rationalized by performing control experiments in the predominant presence of each reactive species formed by radiolysis of water, and the mechanistic pathway scenario was postulated on these bases.

Full text of DOI:10.3390/molecules24173143

molecules
Article
Converging Fate of the Oxidation and Reduction
of 8-Thioguanosine
Katarzyna Taras-Goslinska 1,†, Fabrizio Vetica 2,3,† , Sebastián Barata-Vallejo 3,4
,
Virginia Triantakostanti 3, Bronisław Marciniak 1,5 and Chryssostomos Chatgilialoglu 2,3,5,
*
1
Faculty of Chemistry, Adam Mickiewicz University, Wieniawskiego 1, 61-712 Poznan´, Poland
R&D Laboratory, Lipinutragen srl, Via Piero Gobetti 101, 40129 Bologna, Italy
ISOF, Consiglio Nazionale delle Ricerche, Via Piero Gobetti 101, 40129 Bologna, Italy
Departamento de Quimíca Organíca, Facultad de Farmacia y Bioquimíca, Universidad de Buenos Aires,
Junin 954, RA-1113 Buenos Aires, Argentina
2
3
4
5
Center of Advanced Technologies, Adam Mickiewicz University, 61-712 Poznan´, Poland
Correspondence: chrys@isof.cnr.it; Tel.: +39-051-639-8309
*
†
These authors contributed equally to the work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Derek J. McPhee
Received: 2 August 2019; Accepted: 28 August 2019; Published: 29 August 2019
Abstract: Thione-containing nucleobases have attracted the attention of the scientific community
for their application in oncology, virology, and transplantology. The detailed understanding of the
reactivity of the purine derivative 8-thioguanosine (8-TG) with reactive oxygen species (ROS) and
free radicals is crucial for its biological relevance. An extensive investigation on the fate of 8-TG
under both reductive and oxidative conditions is here reported, and it was tested by employing
steady-state photooxidation, laser flash photolysis, as well as γ-radiolysis in aqueous solutions.
The characterization of the 8-TG T1 excited state by laser flash photolysis and the photooxidation
experiments confirmed that singlet oxygen is a crucial intermediate in the formation of the unexpected
reduced product guanosine, without the formation of the usual oxygenated sulfinic or sulfonic
acids. Furthermore, a thorough screening of different radiolytic conditions upon γ-radiation afforded
the reduced product. These results were rationalized by performing control experiments in the
predominant presence of each reactive species formed by radiolysis of water, and the mechanistic
pathway scenario was postulated on these bases.
Keywords: photolysis; laser flash photolysis; γ-radiolysis; singlet oxygen; nucleosides; free radicals;
reaction mechanism
1. Introduction
The search for new purine and pyrimidine derivatives of biological significance has been conducted
by many research groups [ ]. The analogues of purine bases of nucleic acids containing a sulfur
1,2
atom, such as mercaptopurine, 6-thioguanine, or azathioprine, have been applied in medicine for
many years in the treatments of cancer, in particular leukemia in children, viral diseases, and in
transplantology [
drugs for a long time becomes much more sensitive to sunlight, and the chances of skin cancer
development in these patients increase [ ]. A small structural modification involving the attachment
of a thione group to the purine ring system results in a decrease in energy of the electronic excited states,
leading to absorption of long-wavelength light, including the UV-A range [ ]. Moreover, thiocarbonyl
compounds in excited states are highly reactive [ ]. Therefore, thio-derivatives of guanine present in
3–5]. However, it has been recently evidenced that the skin of patients taking these
6,7
8
9
the chains of nucleic acids can take part in photochemical transformations or at least can initiate them.
The photochemical processes upon excitation of thioguanosine chromophore in DNA include oxidation
Molecules 2019, 24, 3143; doi:10.3390/molecules24173143
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3143
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of DNA bases and/or proteins by 1O2 or other reactive oxygen species (ROS), photo cross-linking of
DNA strands, and generation of reactive radicals that can modify the components of nucleic acids
or proteins. In the photochemical studies of 6-thioguanosine (6-TG), most attention has been paid
to the oxidation reactions, as they are assumed to be mainly responsible for the adverse biological
effects [10
undergoes Type I and Type II photooxidation processes and generates ROS, including singlet oxygen
1O2 or radicals, harmful to biological systems [12
13]. After selective excitation, 6-TG built in DNA
,11]. It has been reported that in aerated aqueous solutions, after absorption of UV-A, 6-TG
,
generates singlet oxygen 1O2 through energy transfer from the excited electronic state of the 6-TG
molecule to the ground state oxygen molecule. Other reactive individuals formed in the presence of
oxygen include radicals generated in the process of electron transfer or charge–transfer complexes
between the triplet state of 6-TG and 3O2 [8,12].
Guanine substituted at the C8 position with either a bromine or thiol group is a potent adjuvant [14].
As an approach to reveal the photooxidation mechanism, we have started the investigations on the
properties of 8-thioguanosine (8-TG, 1) excited states by steady-state and time-resolved techniques.
The electronically excited 8-TG molecule in the presence of oxygen generates reactive species, including
singlet oxygen 1O2 or radicals.
On a structural point of view, the presence of a sulfur atom at the C8 position in 8-TG results in
the establishment of a tautomeric equilibrium between the thione and the thiol form. Equilibrium of
mercapto-azoles has attracted interest in both experimental and theoretical chemistry because of the
relevance of this molecule in biological applications. Theoretical studies on 2-mercaptobenzimidazole,
with a similar heterocyclic scaffold to 8-TG, revealed that the equilibrium favors the thione form
(tautomerization energy thiol/thione is
would favor the thione form as well (Scheme 1).
−
34.2 kJ/mol) [15]. Thus, it is reasonable to consider that 8-TG
Scheme 1. Tautomeric equilibrium of 8-thioguanosine (8-TG).
Therefore, 8-thioguanosine could potentially have a similar reactivity as the thioureas, specifically
cyclic thioureas. The thiourea scaffold has been widely investigated over the years for its applications in
synthetic organic chemistry, industry [16], medicinal chemistry, and agrochemistry [17], and as an ROS
scavenger [18,19]. Because of the highly applicability, several research groups have been interested in the
redox chemistry of thioureas and have reported detailed mechanistic studies for these transformations.
The reactivity of thioureas with hydrogen peroxide [20], common reducing agents such as K and
NaBH4 [21], singlet oxygen [22], and upon pulse radiolysis in both reducing and oxidizing conditions
have been studied [23–29], outlining multiple mechanistic scenarios.
With these premises, in this study we report our investigation on the steady-state photooxidation
and laser flash photolysis of 8-TG, as well as its
γ-radiolysis in aqueous solution, under both reductive
and oxidative conditions, highlighting an unusual and converging product formation.

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2. Results and Discussion
2.1. Structural Characterization of 8-Thioguanosine (8-TG)
Initially, we decided to register NMR spectra in order to define which tautomeric form of 8-TG
is favored. The proton NMR in DMSO-d6 shows 2 different broad signals at 11.13 and 12.63 ppm,
corresponding to NH from amide and thioamide moieties (Figures S1–S3). Moreover, both peaks
exchange after adding D2O. These results, combined with the previously reported theoretical studies
(vide supra), suggest that the equilibrium favors the thione form.
Additionally, we decided to perform a UV titration of aqueous solutions of 8-TG to calculate the
pKa value. Using 12 different 0.1 mM solutions of 8-TG, each at a different pH (using NaOH/phosphoric
acid buffers), we recorded the absorption values and we plotted the absorption versus pH of the
solution (Figure 1). The resulting data were fit into sigmoid curves, and the inflection points were
calculated. The titration curve showed 2 pKa values: pKa1 = 1.87 and pKa2 = 7.88, which presumably
correspond to NH2 protonation and deprotonation of the amide moiety, respectively [30,31].
Figure 1. UV-pH titration of 8-TG.
2.2. Steady-State Photochemistry
Direct excitation of 8-TG in air-equilibrated aqueous solution with
monochromatic radiation at 313 nm leads to disappearance of the intense absorption band
(λmax = 284 nm, ε = 18,700 M
−1 cm−1) of the substrate and increase in absorption in the range of
λ > 300 nm light or
·
max
240–260 nm (Figure 2a). The spectral changes are compatible with the expectation that the thiocarbonyl
group is engaged in the photoreaction. The HPLC analysis revealed 88.5% conversion of the substrate
and a formation of guanosine (Guo,
with a chemical yield of 89% based on the substrate reacted (Scheme 2).
Guo ( ) was found to be the major photoproduct under very low (<15%) conversion of 8-TG
2) as the major photoproduct (Figure 2b). The product was formed
2
as well (Scheme 2). The UV absorption spectra recorded during the irradiation and chromatogram
of the irradiated solution are presented in Figure 2. The product concentration profile showed
that it was present at very low substrate conversion (Figure 3a). Therefore, it did not arise from
secondary photochemistry.

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(a)
(b)
Figure 2. (a) UV spectra of 8-TG (c = 1.8 × 10−4 M) in air-equilibrated aqueous solution before (black)
and after 240 min. Irradiation at
and after irradiation (black).
λ = 313 nm, (b) Chromatogram of the solution of 8-TG before (red)
Scheme 2. The formation of Guo (2) upon excitation of 8-TG (1).
(a)
(b)
Figure 3. (a) The concentration of 8-TG (red) and Guo (blue) as a function of the irradiation time in
air-equilibrated aqueous solution. ( ) Rates of substrate disappearance upon radiation at > 300 nm of
b
λ
8-TG in air-equilibrated solutions with different solvents.
In contrast to the efficient photochemistry in the presence of atmospheric oxygen, the disappearance
of the substrate was slowed down by a factor of 10 when aqueous solution was purged by argon
for 15 min prior to irradiation (Figure 3b). Several products were observed, but guanosine was not
identified. Thus, the presence of oxygen is required for the photochemical transformation of 8-TG
to Guo, suggesting that the observed transformation is a photooxidation reaction. The nature of the
solvent has an impact on the rate of 8-TG disappearance (Figure 3b). A higher rate was observed in
aerated organic solvents. Moreover, the photochemical reaction rate of 8-TG in D2O increased twofold
as compared to the rate determined in aqueous (H2O) solution.
In general, two mechanisms of photochemical oxidation of organic compounds can be
distinguished: a mechanism (type II) with the involvement of singlet oxygen (1O2), a highly reactive
oxygen molecule in its excited electronic state, and a nonsinglet oxygen mechanism (type I) [7,32].

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In order to establish which mechanism was operating in the case of 8-TG, the compound was
subjected to a reaction with 1O2. Methylene blue (MB) and rose bengal (RB) are very effective sensitizers
of 1O2 upon visible light irradiation in aerobic conditions [33]. Figure 4 shows the absorption spectra
of a mixture of MB and 8-TG before and after irradiation (λ > 400 nm) and a chromatogram of the
irradiated mixture. Guo was found to be a major product in both MB- and RB-sensitized reactions of
8-TG, with chemical yields of 57% and 54%, respectively.
(a)
(b)
Figure 4. (a) UV absorption spectra recorded in the course of radiation of methylene blue (MB) and
8-TG with λ > 400 nm; (b) Chromatogram of the irradiated mixture.
The formation of Guo upon excitation of 8-TG with
dyes (
λ = 313 nm light and in a reaction sensitized by
λ
> 400 nm) suggests that the reactive oxygen species, 1O2, is involved in the direct photooxidation
of 8-TG (Scheme 3a). The generation of 1O2 by 8-TG, like in the case of 6-TG, may occur via an energy
transfer from the 8-TG triplet excited state (T1) to the oxygen molecule in the electronic ground state.
Scheme 3.
(a) Photochemical oxidation of 8-TG and photosensitized generation of singlet oxygen;
(b) Proposed mechanistic pathway of the 1O2-promoted desulfurization of 8-TG.

Molecules 2019, 24, 3143
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The desulfurized substrate is an unexpected photoproduct obtained from photooxidation of
heteroaromatic thiocarbonyl compounds [34 35]. The photoproduct molecules identified so far always
,
contained oxygen atom(s). The major photoproducts were: ketones, sulfines, and, in the case
of enolizable thiones, sulfinates and sulfonates. For example, the photoproducts obtained from
6-thioguanine, under experimental conditions identical to that applied for 8-TG, have been identified as
guanine 6-sulfinate, guanine 6-sulfonate, and guanine as a minor photoproduct [36]. Comprehensive
theoretical and experimental investigations of 6-TG photoxidation performed by Zou et al. have shown
that all stable isolated photoproducts, most likely, derive from a common, unstable peroxy intermediate
6-(SOOH)-G [37]. 6-(SOOH)-G is suggested to be a primary product of the 1O2 attack on sulfur atoms
in the 6-TG molecule.
Oxygenated sulfur compounds, like sulfinic and sulfonic acids, as well as the carbonyl analog
of 8-TG, have not been detected. Considering what has been previously described for 6-TG and the
proposed mechanism hypothesized for thiourea in the presence of singlet oxygen [22], we propose
1
that a similar [2 + 2] attack of O2 on the thione moiety would lead to a thioperoxyl radical via
4-membered-ring opening. Subsequently, this unstable intermediate could, instead of proceeding
to an oxygenated sulfur compound, evolve to form a carbene, leading afterwards to guanosine (
(Scheme 3b).
2)
2.3. Laser Flash Photolysis
It has been established that the excited triplet state plays a key role in the generation of singlet
oxygen. That is why the characterization of this state, configuration, radiative and radiation-less
pathways of deactivation, and yield of singlet oxygen generation is so important [38].
The properties of the lowest energy excited states of 8-TG have been studied by laser flash
photolysis. In argon-saturated aqueous solution, only transient absorption of the excited triplet state
(T1) of 8-TG can be observed in nanosecond and microsecond time scales. Transient absorption spectra
of 8-TG in Ar-saturated aqueous solution are shown in Figure 5a. Immediately after the laser, a broad
spectrum extending over 330–750 nm was observed. From these kinetic traces, it was found that the
decay of T1 was independent of the monitoring wavelength, suggesting that the two bands can be
assigned to a single excited species. Single exponential fits were made for the transient absorption
decays measured for a series of 8-TG solutions having concentrations in the range of 0.05–0.5 mM.
The determined rate constants were then used in the Stern–Volmer plot. Under these conditions, the T1
state of 8-TG was relatively long-lived. The intrinsic (concentration-independent) T1 state lifetime,
τ0 = 3.6 µs, was determined from the Stern–Volmer plot (Figure 5c). The T1 state is quenched rapidly
by the ground state O2 molecules with a rate constant kQ = 5
solution). The kinetic traces of the T1 state of 8-TG in argon-saturated and air-equilibrated aqueous
solution, respectively, determined by monitoring the absorption decay at = 620 nm are presented
×
109 M−1
·
s
−1 (in air-equilibrated aqueous
λ
in Figure 5b. The primary photochemical processes in aqueous solution are triplet state formations,
as shown by flash photolysis.
Since 8-TG (T1)* is quenched by dissolved molecular oxygen, a sensitization reaction between
8-TG* and molecular oxygen should occur [39]. In the presence of oxygen, radicals can also be formed
in the process of electron transfer. In particular, evidence for presence of the 8-TG radical or radical
cation was sought in transient absorption measurements. The transient absorption spectrum of 8-TG
obtained under aerated conditions is identical to the spectrum recorded in the Ar-saturated solution.
However, the compared decay dynamics of the T1 state at 340 nm shows the appearance of a new
transient. We observed a long-lived transient (
λ
= 340 nm) formed in reaction of 8-TG with oxygen.
max
Based on our experience and literature reports we can assume that the observed species is a radical or
radical cation. (Figure 6). The transient was found to disappear 35.0 s after the laser pulse, it was not
max = 350 nm) was in the range where guanosine
µ
quenched by oxygen, and the recorded spectrum (
radicals were previously observed [31].
λ

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(a)
(b)
(c)
Figure 5. (a) Transient absorption spectrum of 8-TG (0.62 mM) in argon-saturated aqueous solution
at various delay times ( = 266 nm, 3 mJ). Insert: kinetic traces of the 620 nm absorbance decay
λ
exc
and 310 nm the ground state depopulation of 8-TG in argon-saturated aqueous solution; (
traces of the 620 nm absorbance decay in argon-saturated and air-equilibrated aqueous solution; (
Stern–Volmer plot of reciprocal of triplet lifetime vs. concentration of 8-TG in argon-saturated solution.
b) Kinetic
c)
(a)
(b)
Figure 6. (a) Transient absorption spectrum of 8-TG (0.62 mM) in air-equilibrated aqueous solution
at various delay times ( = 266 nm, 3 mJ). ( ) Kinetic trace of the 340 nm absorbance decay in
λ
exc
b
air-equilibrated aqueous solution.
For 8-TG, the radical processes have not been observed so far, but they are well known for
guanine [40]. Molecular oxygen is known to act as an electron acceptor whether it is in its ground state
or in its excited singlet state [41]. Both processes are presented below with the participation of 8-TG in
its T1 excited state or in its ground state (S0).
•
8-TG(T1) + 3O2 → 8-TG+• + O2
,
(1)

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•−
8-TG(S0) + 1O2 → 8-TG+• + O2
,
(2)
2.4. γ-Radiolysis of 8-TG in Aqueous Solutions
Radiolysis of neutral water leads to the reactive species eaq−, HO•, and H•, together with H+ and
H2O2, as shown in Equation (3). The values in brackets represent the radiation chemical yield (G) in
µmol·J−1 [42].
H2O + γ-irr. → eaq−(0.27), HO•(0.28), H•(0.06), H+(0.06), H2O2(0.07)
(3)
Having discovered the unexpected reductive desulfurization of 8-TG in oxidative conditions, our
investigation proceeded with the -radiolysis of in the presence, initially, of all the reactive species
γ
1
generated by radiolytic decomposition of water: eaq−, HO•, and H•. Irradiations were performed at
room temperature using a 60Co-Gammacell at different doses. The exact absorbed radiation dose was
determined by employing the Fricke chemical dosimeter, by taking G(Fe3+) = 1.61 µmol·J−1 [43].
We investigated this reaction by detailed product studies in a dose-dependent experiment,
irradiating 6 different independent samples (each 0.196 mM of 1) for 100 Gy dose intervals, until
achieving a total dose of 500 Gy. The reaction mixtures were monitored by HPLC-UV (254 nm) analysis,
and the chromatograms were plotted in Figure 7a. In the dose course, we could observe consistent
consumption of the starting material
main product (blue peak, retention time (RT) = 9.8 min). Mass spectra of this product showed the
mass of guanosine , also confirmed by comparison with commercially available guanosine as well as
spiking experiments. The product yield (mol )
kg−1) divided by the absorbed dose (1 Gy = 1 J kg−1
1 (red peak, RT = 13.01 min) and simultaneous formation of the
2
·
·
gives the reaction’s chemical yield (G). The G values at each analyzed dose were calculated and plotted
with the dependence of the dose (Figure 7b). The radiation chemical yields extrapolated to the zero
dose are: G (−1) = 0.17 and G (
species (Equaction (3)) of 0.61
0.44 (or 0.51 respectively) of the reacting radicals returned to starting materials, while 0.17 reacted
productively with . Moreover, 70.6% of these radicals (G = 0.12 mol
−1), lead to the formation of
product . Unknown products formed and were visible in the formation of small peaks at RT = 8.6 and
14.45 min.
2
) = 0.12
µ
mol J
−1. Considering the sum of the G values of the reactive
·
µmol
·
J−1 (0.68 if we consider H2O2 contribution), we could observe that
1
µ
·
J
2
(a)
(b)
Figure 7. γ-radiolysis of
Gy
min−1. (
of guanosine (
1
(0.196 mM) in N2-purged water at a natural pH at a dose rate of 1.85
·
a
) HPLC runs of the reactions. The HPLC peaks of
) are highlighted in blue. (
1
are highlighted in red, while the peaks
) and G ( ) ( ) as
2
b
) The chemical irradiation yields G (−1) (
ꢀ
2 ꢀ
a function of the irradiation dose. The line extrapolated to the zero dose leads to the G values reported
in the graph.

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The results obtained in the above
γ
-radiolysis experiment and in the absence of oxidation products,
such as 8-oxo-Guo or the disulfide dimer of 8-TG, underline an unexpected and more complex
mechanistic scenario that is in accordance with the results obtained in the photooxidation experiments.
Therefore, we decided to investigate some aspects of these reactions in more detail.
Before moving forward to the reactive species, we decided to first test the reactivity of
1 with
H2O2, which is also formed by radiolysis of water. Indeed, the reaction of thiourea derivatives with
hydrogen peroxide is a well-known desulfurization process, leading to the reduced product and
SO2 [20]. Therefore, we carried out an experiment outside the Gammacell using an aqueous solution
of 8-TG (0.3 mM) in the presence of 10% H2O2, kept without stirring for 4.5 h, and a time comparable
to 500 Gy if the reaction was done in Gammacell (Figure S12, Supporting Information). As expected,
the formation of Guo (2) as a major product was observed.
2.4.1. γ-radiolysis of 8-TG in Aqueous Solutions under Reductive Conditions
Afterwards, we focused first on the reactivity of eaq− and H• with
1, employing N2-saturated water
solutions at a natural pH. tBuOH (0.25 M) was added to the reaction mixture in order to suppress the
HO• generated from the radiolytic decomposition of water (Equation (4)). In fact, efficient conversion
−
of hydroxyl radicals into the less reactive tert-butyl radicals allows studying the reaction of the eaq
and H• atoms because the presence of tBuOH also suppresses H• radicals, but only slightly since the
reaction rate is more than three orders of magnitude lower (Equation (5)) [42].
HO• + tBuOH → (CH3)2(OH)CH2• + H2O
H• + tBuOH → (CH3)2(OH)CH2• + H2
(k = 6.0 × 108 M−1·s−1
)
(4)
(5)
(k = 1.7 × 105 M−1·s−1
)
From Figure 8a we can infer that the gradual disappearance of 8-TG occurred with the formation
of Guo ( ). Furthermore, no evidence of any side reaction was found, a part from some negligible
2
peaks eluted after the starting material (RT between 14 and 15 min). The G values at each analyzed
dose were calculated and plotted with the dependence of the dose (Figure 8b). The radiation chemical
yields extrapolated to the zero dose are: G (−1) = 0.15 and G (
that all the reacted 8-TG was converted solely to product
in a similar scenario, to the previous experiments without tBuOH.
2
) = 0.14
µ
mol
·
J
−1. These values underline
2
or returned to the starting material and,
(a)
(b)
1 (0.25 mM) in N2-purged water at a natural pH, containing tBuOH (0.25 M)
Figure 8. γ-radiolysis of
at a dose rate of 1.85 Gy
·
min−1. (
) HPLC runs of the reactions. The HPLC peaks of are highlighted in
red, while the peaks of guanosine ( ) are highlighted in blue. (
) The chemical irradiation yields G (−1
) and G ( ) ( ) as function of the irradiation dose. The line extrapolated to the zero dose leads to the
G values reported in the graph.
a
1
2
b
)
(
ꢀ
2 ꢀ

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On the basis of these and the previous results, our mechanistic hypothesis is depicted in Scheme 4.
We postulated that the contribution of H2O2 would proceed, as reported in literature [20], via formation
of the oxygenated intermediate
to product
. On the other hand, H• radicals could add on the thione moiety of 8-TG, forming the
radical , as suggested by previously reported studies [25]. Subsequently, this species could eliminate
HS• radicals, forming the same carbene
formed with H2O2 and achieving Guo ( ). The combined
G values of H• radicals and H2O2 correspond to 0.13 J−1 and the G ( −1; hence,
mol ) = 0.12 mol
C, which eliminated SO2 and generated a carbene, which then led
2
A
B
2
µ
·
2
µ
·
J
they were in accordance with the suggested pathway. However, the reaction of eaq−/H+ should also
potentially contribute to the formation of intermediate A.
O
N
O
N
O
N
H
N
H
N
N
N
H
NH
NH2
NH
NH2
NH
NH2
H
HS
G = 0.06
N
N
R
R
R
HS
A
B
2
or
–H+
pK a = 4
H+
S
eaq
1
G = 0.27
SO2
O
O
N
O
N
H
N
H
H2O2
O
O
N
N
NH
NH2
NH
NH2
NH
NH2
S
H
G = 0.07
N
N
N
N
R
R
R
C
B
2
Scheme 4. Postulated mechanism of desulfurization promoted by H• atoms and H2O2.
Concerning the experiment suppressing HO• radicals, on the basis of the observed similar
G values, we postulated that the reaction could involve H• atoms and H2O2 (Scheme 4) as well.
Indeed, both these species are present in the reaction medium. However, we were interested−to also
−
understand the involvement of eaq in the formation of Guo (
2
). The reaction of 8-TG with eaq could
generate a radical anion, which, upon protonation, could afford the previously discussed radical
formed by the attack of H• atoms. Therefore, the contribution of both H• radicals and/or eaq−/H+
would provide the formation of Guo (2) via elimination of HS• and formation of carbene B.
With these premises, to confirm whether the mechanistic pathway of this desulfurization proceeded
via heterolytic bond cleavage or by thiyl radical formation, we performed the same three experiments
in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (2 mM) on
the basis of our extensive experience in lipid cis/trans isomerization catalyzed by thiyl radicals
(Scheme 4) [44–46]. In fact, if the mechanism involves the elimination of sulfhydryl radicals, we should
observe isomerization, while no isomerization should occur if SO2 is produced. After 4.5 h reaction
time or 500 Gy irradiation, the phospholipids were extracted, transesterified to the methyl esters in
methanolic KOH (0.5 M) solution, and analyzed via gas chromatography (GC) (for details see 3.6–3.8).
GC analysis of the fatty acid methyl esters revealed that no isomerization occurred in the reaction
with H2O2, supporting our hypothesis, which involves the formation of SO2 (Figure S12). Conversely,
in the
of HS• radicals (Figure S7). Moreover, the reaction performed under reductive conditions achieved the
desulfurized product while providing isomerization of the oleic double bonds up to 13.7% (Figure S8).
γ-radiolysis experiment we observed 4.5% of isomerization, supporting the postulated elimination
2

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2.4.2. The Role of HO• Radicals and Other Oxidants
In a subsequent experiment, we deemed it proper to study the reaction of
1
with HO• radicals.
In doing so, we performed radiolysis of
1
in N2O-saturated water on the basis of the previously
−
discussed transformation. In N2O-saturated solution at a natural pH (
≈
0.02 M of N2O), eaq are
efficiently transformed into HO• radicals via Equation (6) (k = 9.1
×
109 M
s
−1· −1), affording G
(HO•) = 0.55 µmol·J−1
;
that is, HO• and H• account for 90% and 10%, respectively, of the reactive
species [42].
eaq− + N2O + H2O → HO• + N2 + HO−
(k = 9.1 × 109 M−1·s−1
)
(6)
Solutions of
and the results are plotted in Figure 9a. Also in this experiment, we observed a gradual consumption
of (red peaks) with concurrent formation of the reduction product ( , blue) despite the oxidative
conditions. At a dose of 600 Gy the product yield was 14%, while the conversion was 54%. However,
in this case, we observed the formation of 2 additional peaks, one with RT of 9 min and the
other 14.5 min, which could explain the lower conversion measured. The radiation chemical yields
extrapolated at zero dose from the plots in Figure 9b were: G (−1) = 0.16 and G ( ) = 0.12 mol
J−1
Assuming that these results derive as before from the contribution of H• radicals and H2O2, since G
(HO•) is 0.55 −1, the arising question is what happened to HO• radicals since we did not observe
mol
further consumption of the substrate 1.
1 (0.3 mM) were irradiated and stopped at intervals of 100 Gy, up to 500 Gy,
1
2
≈
≈
2
µ
·
.
µ
·J
(b)
(a)
Figure 9. γ-radiolysis of
1
(0.3 mM) in N2O-purged water at a natural pH at a dose rate of 1.85
are highlighted in red, while the peaks
) and G ( ) ( ) as
Gy ) HPLC runs of the reactions. The HPLC peaks of
min−1. (
of guanosine ( ) are highlighted in blue. (
·
a
1
2
b
) The chemical irradiation yields G (−1) (
ꢀ
2 ꢀ
function of the irradiation dose. The line extrapolated to a zero dose leads to the G values reported in
the graph.
To answer this question, we decided to employ different oxidant species generated from the
conversion of hydroxyl radicals (from Equation (6)), depending of the type of additive present •in−
an N2O-saturated solution. In particular, we decided to test the reactivity of 1 in the presence Br2
•−
and N3•, formed via Equations (7) and (8) from KBr (0.1 M) and NaN3 (0.1 M) (respectively G (Br2
and G (N3•) = 0.55 µmol·J−1).
)
•
HO• + N3− → N3 + HO−
(k = 1.2 × 1010 M−1·s−1
(k = 1.1 × 1010 M−1·s−1
)
(7)
(8)
HO• + 2Br− → Br2•− + HO−
)

Molecules 2019, 24, 3143
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In Figure S5 the HPLC-UV runs for the dose-dependent experiments in the presence of N3• are
depicted. The main product formed was the reduced product guanosine ( ) at a 33.6% yield and 95.7%
conversion after 600 Gy irradiation. From the dose course studies, we could observe the gradual
2
consumption of
of N3 radicals reacted with
product. Interestingly, the same behavior was observed by using Br2 , which measured similar G
values and an identical difference between G (−1) and G (2) of 0.07 µmol·J−1 (Figure S4).
With these results in hand, we were interested in understanding the involvement of these oxidant
1 while 2 consistently formed. The radiation chemical yields highlight that about half
•
1
in a productive way, while 66% of these contributed to formation of the
•−
species in the overall desulfurization of 8-TG (1). In fact, in all the reactions we could observe similar
results to the ones obtained in the previous experiments in terms of G values, while no further
consumption of the substrate was achieved via side product formation. On the basis of our strong
background of radical chemistry of guanosine derivatives [31
radicals could perform hydrogen abstraction predominantly on the exocyclic NH2 group [48
leading to the radical intermediate (Scheme 5). This species could undergo tautomerization to the
more stable radical . We proved that this tautomeric equilibrium was not affected by substitution at
,47–
52], it was established that HO•
–
50],
D
E
the 8-position of the guanine moiety with H, Br, or alkyl group, and it occurred at a constant of about
3 × 104 s−1 [50]. Hence, we could assume that this could be the case as well for 8-TG-derived radical
D.
Once the tautomeric radical
E formed, this could lead back to the starting material 1. In fact, this has
been reported for similar intermediates for the other studied guanosine derivatives, which resulted in
regeneration of the substrate with a still unclear mechanism [31,52]. This pathway could explain the
absence of further consumption of 8-TG in the pred•o−minant presence of HO• radicals. Additionally,
•
the intermediate
E
could be directly formed by Br2 and N3 and provide regeneration of
1
,
in the
51 52].
same fashion, explaining the comparable results in the presence of these two radical species [31
,
•−
Scheme 5. Hypothesized mechanism of the reaction between HO•, Br2 and N3•, forming radical
intermediates and leading back to the substrate.
Subsequently, we carried out all these experiments under oxidative conditions in the presence
of POPC liposomes, to check whether the contribution of H• atoms could lead to the formation of
thiyl radicals in this case as well. In all the reactions, we could observe the promotion of cis/trans
isomerization of the oleic moiety between 2.1–7.9% (Figures S9–S11), confirming that the contribution
of the oxidant species leads to the regeneration of substrate 1.
3. Materials and Methods
3.1. Materials
Unless otherwise noted, all commercially available compounds were used without further
purification. Solvents were purchased at HPLC grade and used without further purification. Water was
purified with a Millipore system. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was
purchased from Larodan Inc. (Solna, Sweden); chloroform, methanol, diethyl ether, and n-hexane
(HPLC grade) were purchased from Baker (Dover, NJ, USA). 8-TG was purchased from Santa Cruz

Molecules 2019, 24, 3143
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Biotechnology Inc. (Dallas, TX, USA). Guanosine, D2O, and DMSO-d6 were purchased from Sigma
Aldrich (St. Louis, MO, USA).
3.2. UV-Vis Absorption Spectra
The UV absorptions for the determination of the pKa were recorded with a UV-vis spectrometer
Agilent Carey 100 (Santa Clara, CA, USA). Using 12 different 0.1 mM solutions of 8-TG, each at a different
pH (using NaOH/phosphoric acid buffers), we recorded the absorption values and we plotted the
absorption versus pH of the solution. The resulting data were fit into sigmoid curves, and the inflection
points were calculated. Figure 1 was prepared with Graphpad Prism 8.0 (San Diego, CA, USA).
All other UV spectra were recorded at room temperature with a Cary300Bio spectrophotometer.
3.3. HPLC-MS Analysis
HPLC-MS analyses were performed with an Agilent 1260 Infinity II HPLC system equipped
with a Fortis 5
µ
m UniverSil C18 column (250
×
4.6 mm) using, as eluent, 0.01% formic acid in
water with 0–55% gradient of acetonitrile, coupled with an InfinityLab single quadrupole liquid
chromatography/mass selective detector (LC/MSD) and with a diode array detector set at 254 nm.
Quantitative studies of the performed reactions were done by multiple point calibration curves
(6 points) in HPLC-MS equipped with a Fortis 5
eluent, 0.01% formic acid in water with 0–55% gradient of acetonitrile (injection volume 5
µ
m UniverSil C18 column (250
×
4.6 mm) using, as
L, flow
µ
rate 0.7 mL/min, detection at 254 nm). Elution method: (A = H2O + 0.01% formic acid, B = ACN)
A:B = 95:5 1 min, 45:55 at 11 min and held for 5 min, 45:55 at 15 min, back at 95:5 at 18 min, and held
for 5 min. R2 = 0.99998 8-TG, 1.0 Guo. Retention times: 8-TG 13.02 min, Guo 9.8 min.
3.4. Steady-State Irradiation
Solutions (2.5 mL) of 8-TG (1.8
irradiated using a 200 W high-pressure mercury lamp equipped with a pyrex (
an interference filter ( = 313 nm). For irradiation in the absence of oxygen, solutions were deaerated by
×
10-4 M) in ACN or H2O in a quartz cell (l = 1 cm) were
> 300 nm) or
λ
λ
bubbling with argon; otherwise, the solutions were irradiated in an open-to-air cell (air-equilibrated).
In dye-sensitized photoreactions, solutions containing methylene blue or rose bengal (c = 0.05 mM)
and 8-TG (1.8 × 10−4 M) were irradiated in air-equilibrated ACN or H2O solutions at >400 nm.
The photoreactions were monitored by measuring the absorbance spectrum. The concentrations of
substrate and photoproducts were determined by HPLC analysis. The photoproduct guanosine was
identified by a comparison of the HPLC peak retention time, UV–VIS absorption, and MS spectra with
a standard reference.
3.5. Laser Flash Photolysis (LFP) Experiments
The set up for the nanosecond laser flash photolysis (LFP) experiments and its data acquisition
system have previously been described in detail [41]. LFP experiments employed a pulsed Nd:YAG
laser (λ = 266 nm, 3 mJ, 3–9 ns) for excitation. Transient decays were recorded at individual wavelengths
by the step-scan method with a step distance of 10 nm in the range of 320–800 nm as the mean of
10 xenon-lamp pulses.
3.6. Experiments in Gammacell
Irradiations were performed at room temperature (22
±
2 ◦C) using a 60Co-Gammacell at different
dose rates. The exact absorbed radiation dose was determined with the Fricke chemical dosimeter,
by taking G(Fe3+) = 1.61 µmol·J−1 [43].
In the radiation experiments, 4 mL vials equipped with a rubber septum were used. Two milliliters
of freshly prepared stock solutions (0.09 or 0.3 mM) of 8-TG were added together with specific amounts
of additives, depending of the experiments (see above). Afterwards, the solution was degassed with

Molecules 2019, 24, 3143
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either N2 or N2O for 10 min via a cannula, sealed with parafilm, and irradiated in a Gammacell.
After the irradiation time, the reaction mixtures were injected in HPLC-MS without further workup.
For kinetic experiments, different reactions were prepared from a single mother solution, and then
each one was stopped at a different dose. Figures were prepared with the software Graphpad Prism 8.0
and OriginLab.
For the experiments in the presence of POPC liposomes, the workup of the reactions was done
as follows: 2:1 chloroform/methanol (5
×
4 mL) was added to the reaction mixture according to the
Folch method [53]. The organic layer was collected and dried over anhydrous Na2SO4 then evaporated
under vacuum to dryness. One milliliter of 0.5 M solution of KOH in methanol was added to each
sample for transesterification of the fatty acid containing esters, which was performed by stirring
for 30 min at room temperature. The methanolic reaction mixture was quenched with brine (1 mL),
then the extraction was repeated with n-hexane (3
dried over anhydrous Na2SO4, and evaporated to dryness. The fatty acid methyl ester (FAME) residue
×
2 mL), and the organic phases were collected,
of each sample was dissolved in 50 µL of n-hexane, and 1 µL was injected for GC analysis.
3.7. Preparation of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) Liposomes
Large unilamellar vesicles by extrusion technique (LUVETs) were prepared using the
hydration-extrusion technique [54]. Briefly, POPC (76 mg; 0.1 mmol) was dissolved in CHCl3/MeOH
2:1 until a clear lipid solution was obtained. The organic solvent was removed using a rotary evaporator
to yield a homogeneous lipid film on the sides of a round-bottom flask. The lipid film was thoroughly
dried to remove residual organic solvent by placing the vial or flask on a vacuum pump for 1 h.
The dried lipid film was left to hydrate for 30 min in PBS and then vortexed for 10 min until a milky
monophasic solution containing multilamellar vesicles (MLVs) was obtained. Once a stable suspension
was formed, MLVs were downsized to LUVETs using a LiposoFast hand extrusion device (Avestin
Inc., Ottawa, ON, Canada) equipped with 100 nm pore size polycarbonate filters through which the
lipid suspension was passed 19 times, controlling the temperature with a heat block when required.
The resulting suspension was used in the irradiation experiments.
3.8. GC Analysis of Fatty Acid Methyl Esters
Fatty acid methyl esters (FAMEs) were analyzed by GC (Agilent 6850, Milan, Italy), using the split
mode (50:1), equipped with a 60 m
×
0.25 mm
×
0.25 µm (50% cyanopropyl)-methylpolysiloxane column
(DB23, Agilent, USA), and a flame ionization detector with the following oven program: temperature
started at 165 ◦C, held for 3 min, followed by an increase of 1 ◦C/min up to 195 ◦C, held for 40 min,
followed by a second increase of 10 ◦C/min up to 240 ◦C and held for 10 min. A constant pressure
mode (29 psi) was chosen with helium as the carrier gas. FAMEs were identified by comparison with
authentic samples, and chromatograms were examined as described previously.
3.9. NMR Analysis of 8-TG
NMR spectra were recorded at ambient temperature on a Varian 500 MHz spectrometer. D2O and
DMSO-d6, purchased from Sigma Aldrich (St. Louis, MO, USA), were used as solvents.
4. Conclusions
In conclusion, we reported a detailed study on the reactivity of biologically relevant
8-thioguanosine under oxidative and reductive conditions by using photooxidation, laser flash
photolysis, and γ-radiolysis.
Direct excitation (λ > 310 nm) of 8-thioguanosine in aerated aqueous solution gave guanosine
instead of oxidized sulfur derivatives (purine sulfonic and sulfinic acids). It appears reasonable
1
to postulate that 8-TG transformation under direct irradiation occurs with intermediacy of O2.
The properties of the lowest energy excited state of 8-TG (T1) have been studied by laser flash
photolysis, and it was subsequently established that this excited T1 state could act as a sensitizer,

Molecules 2019, 24, 3143
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while in its ground state it could act as an acceptor of 1O2. Guanosine, formally a reduction product of
8-TG, is a rather unexpected photoproduct obtained from the photooxidation reaction of thiocarbonyl
compounds, and singlet oxygen is a key intermediate in the desulfurization of the compound studied.
Subsequent investigations on the γ-radiolysis of 8-TG highlighted a complex mechanistic scenario,
which was studied by evaluating the effects of the predominant presence of each reactive specie•s
derived by the radiolysis of water. The presence of oxidant species such as HO• radicals, Br2•−, and N3
resulted, supposedly, in the regeneration of substrate 1. Conversely, control experiments in the presence
of POPC liposomes and reductive conditions displayed the possible involvement of H• atoms, H2O2,
and/or eaq−/H+ in the desulfurization of 8-TG by the generation of thiyl radicals and the simultaneous
formation of the desulfurized product 2.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/17/3143/s1,
1
Figure S1: 1H NMR spectrum of 8-TG; Figure S2: Zoomed•−H NMR spectrum of 8-TG; Figure S3: 1H NMR
•
spectrum of 8-TG after adding D2O; Figure S4: Reaction of Br2 with 8-TG; Figure S5: Reaction of N3 with 8-TG;
Figure S6: Reaction of 8-TG in the presence of H2O2 outside Gammacell; Figure S7: Reaction of HO•, H•, and eaq
−
−
with 8-TG in the presence of POPC liposomes; Figure S8: Reaction of eaq with 8-TG in the presence of POPC
liposomes; Figure S9: Reaction of HO• with 8-TG in the presence of POPC liposomes; Figure S10: Reaction of
•−
•
Br2 with 8-TG in the presence of POPC liposomes; Figure S11: Reaction of N3 with 8-TG in the presence of
POPC liposomes; Figure S12: Reaction of H2O2 with 8-TG in the presence of POPC liposomes.
Author Contributions: K.T.-G. and F.V. contributed equally to this work; K.T.-G., B.M., C.C., and F.V designed
the study; C.C. and B.M. coordinated the work; C.C., F.V., and K.T.-G. wrote the paper; C.C., S.B.-V., F.V., B.M.,
and K.T.-G. discussed the project; F.V. and S.B.-V. performed the experiments in Gammacell and collected the
worked-up samples; K.T.-G. performed the time steady-state photooxidation and laser flash photolysis; F.V.
performed the HPLC-MS analyses of the experiments in Gammacell; C.C., S.B.-V., and F.V. discussed the HPLC-MS
results; V.T. performed the GC analyses of the experiments with liposomes; C.C., S.B.-V., F.V., and K.T.-G. prepared
all the figures. All the authors reviewed the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors thankfully acknowledge the contribution of Graz˙yna Wenska.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
8-TG
8-thioguanosine
6-TG
6-thioguanosine
ROS
reactive oxygen species
guanosine
fatty acid methyl ester
high performance liquid chromatography mass spectrometry
gas chromatography
Guo/G
FAME
HPLC-MS
GC
POPC
MB
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Methylene Blue
RB
Rose Bengal
8-oxo-G
8-oxo-guanosine
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