A Combined Experimental and Theoretical Approach to the Photogeneration of 5,6-Dihydropyrimidin-5-yl Radicals in Nonaqueous Media

Article abstract of DOI:10.1021/acs.joc.6b00314

The chemical fate of radical intermediates is relevant to understand the biological effects of radiation and to explain formation of DNA lesions. A direct approach to selectively generate the putative reactive intermediates is based on the irradiation of photolabile precursors. But, to date, radical formation and reactivity have only been studied in aqueous media, which do not completely mimic the microenvironment provided by the DNA structure and its complexes with proteins. Thus, it is also important to evaluate the photogeneration of nucleoside-based radicals in nonaqueous media. The attention here is focused on the independent generation of 5,6-dihydropyrimidin-5-yl radicals in organic solvent through the synthesis of new lipophilic tert-butyl ketone precursors. Formation of 5,6-dihydro-2′-deoxyuridin-5-yl and 5,6-dihydrothymidin-5-yl radicals has first been confirmed by using a new nitroxide-derived profluorescent radical trap. Further evidence has been obtained by nanosecond laser flash photolysis through detection of long-lived transients. Finally, the experimental data are corroborated by multiconfigurational ab initio CASPT2//CASSCF methodology.

Full text of DOI:10.1021/acs.joc.6b00314

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Article
A Combined Experimental and Theoretical Approach to the Photogeneration
of 5,6-Dihydropyrimidin-5-yl Radicals in Non-Aqueous Media
Isabel Aparici-Espert, Antonio Francés-Monerris, Gemma M Rodriguez-Muniz,
Daniel Roca-Sanjuán, Virginie Lhiaubet-Vallet, and Miguel Angel Miranda
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00314 • Publication Date (Web): 18 Apr 2016
Downloaded from http://pubs.acs.org on April 20, 2016
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A Combined Experimental and Theoretical Approach to the
Photogeneration of 5,6ꢀDihydropyrimidinꢀ5ꢀyl Radicals in Nonꢀ
Aqueous Media
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Isabel ApariciꢀEspert†, Antonio FrancésꢀMonerris‡, Gemma M. RodríguezꢀMuñiz†,
Daniel RocaꢀSanjuán‡, Virginie LhiaubetꢀVallet†*, Miguel A. Miranda†*
†Instituto Universitario Mixto de Tecnología Química UPVꢀCSIC, Universitat Politècnica de València, Avda de los
Naranjos s/n, 46022 València, Spain. ‡Instituto de Ciencia Molecular, Universitat de València, P.O. Box 22085, 46071
València, Spain
Corresponding authors: eꢀmail: lvirgini@itq.upv.es, mmiranda@qim.upv.es
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TABLE OF CONTENTS
hν
R = H, λ = 400-420 nm
max
R = CH , λ = 350-400 nm
3
max
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ABSTRACT
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The chemical fate of radical intermediates is relevant to understand the biological effects of radiation
and to explain formation of DNA lesions. A direct approach to selectively generate the putative reactive
intermediates is based on the irradiation of photolabile precursors. But, up to date, radical formation and
reactivity have only been studied in aqueous media, which do not completely mimic the
microenvironment provided by the DNA structure and its complexes with proteins. Thus, it is also
important to evaluate the photogeneration of nucleosideꢀbased radicals in nonꢀaqueous media. The
attention here is focused on the independent generation of 5,6ꢀdihydropyrimidinꢀ5ꢀyl radicals in organic
solvent through the synthesis of new lipophilic tertꢀbutyl ketone precursors. Formation of 5,6ꢀdihydroꢀ
2´ꢀdeoxyuridinꢀ5ꢀyl and 5,6ꢀdihydrothymidinꢀ5ꢀyl radicals have first been confirmed by using a new
nitroxideꢀderived profluorescent radical trap. Further evidence has been obtained by nanosecond laser
flash photolysis through detection of longꢀlived transients. Finally, the experimental data are
corroborated by multiconfigurational ab initio CASPT2//CASSCF methodology.
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INTRODUCTION
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Damage to DNA plays a significant role in the etiology and treatment of diseases. It often occurs
through radical intermediates, which constitute the major family of reactive species generated in nucleic
acids upon exposure to γꢀradiolysis and Fenton conditions. Consequently, the chemical fate of these
species is relevant to understand the biological effects of radiation and to explain formation of DNA
lesions.1ꢀ5 However, elucidating the chemistry of a specific radical is challenging because of the
limitations imposed by the unselective nature of the involved processes. This problem has been tackled
through independent generation of the putative reactive intermediates on isolated nucleosides as well as
on synthetic oligonucleotides. In this context, modified nucleosides have been designed to achieve
selective generation of the purported radicals upon appropriate activation.2,6ꢀ9 Photolabile precursors
have been very useful for studying the reactivity of sugar or nucleobase radicals involved in the
formation of oxidatively generated damage in pyrimidine nucleosides. Lightꢀabsorbing moieties like
isoꢀpropyl or tertꢀbutyl ketone, phenyl sulfide, or phenyl selenide have allowed addressing the issue of
the reactivity of carbon centered radicals at C5, C6, C1´, C2´, C3´, C4´, C5´ or at the thymine methyl
group.6,7,10ꢀ20 Pulse radiolysis, laser flash photolysis, and electron paramagnetic resonance have been run
on aqueous solutions of the pyrimidine or photolabile precursors,21ꢀ23 and transient absorption spectra of
the radicals of interest have been obtained by mathematical treatment of the whole signals usually
corresponding to mixtures of several species.23,24 From these results it has been concluded that 5,6ꢀ
dihydrothyminꢀ5ꢀyl and related radicals show weak (ε ca. 1000 Mꢀ1 cmꢀ1) transient absorption spectra
with broad maxima ranging from 380 nm to 460 nm23,24 and lifetimes of hundreds of microseconds.25
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With this background, the attention here is focused on the independent generation of 5,6ꢀ
dihydropyrimidinꢀ5ꢀyl radicals in nonꢀaqueous medium through the synthesis of new lipophilic tertꢀ
butyl ketone precursors. Photolysis of a tertꢀbutyl ketone photolabile moiety at λ > 300 nm populates
the nπ* excited state, leading to radical formation through a Norrish type I photoreaction.26 Indeed, this
type of process has already been used to establish the chemical fate of thymidine and 2´ꢀdeoxyuridine
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C5 radicals inserted into synthetic oligonucleotides, characterized by strand breaks at the original site
and at the adjacent 5´ꢀnucleotide.10ꢀ13,27 As stated above, the studies reported until now have been
performed in aqueous media. In spite of the large amount of water in mammalian cells, the
microenvironment provided by the DNA structure and its complexes with proteins like histones may not
be fully reproduced by aqueous media, since the pyrimidineꢀderived radical would be embedded into the
complex DNA/RNA system, which constitutes a heterogeneous and flexible environment. Nonꢀaqueous
media would also be of interest for the study of nucleic acid stability within the lipophilic surroundings
supplied by vectors for gene delivery such as liposomes or polymers.28,29 This is especially relevant for
photochemical internalization based on an improved endolysosomal release by photoactivation of a
sensitizer, which could originate DNA/RNA oxidative damage and loss of gene integrity.30,31 In spite of
its interest, information on nucleic acid reactivity in nonꢀaqueous media is essentially lacking, except for
the recently reported oxidation of DNA bases through one electron transfer or hydroxyl radical
attack.32,33
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Hence, the spectroscopic study of lipophilic precursors of 5,6ꢀdihydropyrimidinꢀ5ꢀyl radicals has now
been addressed in acetonitrile as solvent. The main purpose of the work is to obtain transient absorption
data of high value for subsequent timeꢀresolved experiments in nonꢀaqueous solutions, as well as to
provide a new tool for future assessment of the possible modulating effect of organic solvents on the
secondary reactions mediated by peroxyl radicals. The UVꢀVis absorption spectra of the precursors
have been measured in acetonitrile to determine the functional excitation range required to trigger
photodeprotection and to confirm carbonꢀcentered radical formation via its trapping by a nitroxideꢀ
based profluorescent probe. Further evidence supporting generation of the 5,6ꢀdihydropyrimidinꢀ5ꢀyl
radical has been obtained by nanosecond laser flash photolysis. Finally, theoretical calculations based
on the multiconfigurational completeꢀactive space second order perturbation theory//completeꢀactiveꢀ
space selfꢀconsistent field (CASPT2 //CASSCF) protocol corroborate the experimental results.
RESULTS AND DISCUSSION
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The new C5ꢀpivaloyl derivatives 1 and 2 were synthesized following procedures previously described
in the literature for similar compounds (Scheme 1).10,27,34 Briefly, the first step consisted in reduction of
the C5ꢀC6 double bond of uridine or thymidine by hydrogenation using Rh/Al2O3 as catalyst. Next,
C5´and C3´ hydroxyl functions were protected with tertꢀbutyldimethylsilyl group (TBDMS), followed
by protection of nitrogen in position 3 with Nꢀ2ꢀtrimethylsilylethyoxymethyl moiety (SEM) in order to
increase the solubility in organic media. Finally, deprotonation of C5 by lithium diisopropylamide
(LDA) and subsequent reaction with pivaloyl chloride led to the desired compounds.
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The absorption spectra in acetonitrile showed a band centered at 285 or 315 nm for 1 and 2,
respectively (Figure S1 in Supporting Information). This absorption, assigned to the nπ* singlet excited
state of the pivaloyl moiety, is a relevant characteristic because it is linked to the possibility of
performing selective UVA/UVB irradiation of the radical precursor when inserted in DNA.
Scheme 1. Synthetic pathways to obtain the C5ꢀradical precursors derived from uridine and
thymidine
(i)
H2, Rh/Al2O3 in H2O r.t., (ii) Imidazole, TBDMSꢀCl in DMF 0ᵒC → r.t., (iii) iPr2NEt, SEMꢀ
Cl in CH2Cl2 r.t., (iv) LDA, PivaloylꢀCl in THF ꢀ78ᵒC, (v) hν
The photofragmentation of precursors 1 and 2 was first investigated by using nitroxideꢀbased
fluorescent probes, a family of paramagnetic compounds that have been widely used to trap carbonꢀ
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centered radicals.35 Their application as fluorogenic probes has been reported for the study of numerous
biological processes such as redox states detection, reactive oxygen species formation, etc.35,36 This
methodology, which can be employed with complex biological systems, takes advantage of an efficient
intramolecular quenching of the fluorophore emission by the TEMPO moiety. Radical trapping by the
nitroxide leads to a diamagnetic adduct (either alkoxyamine or Nꢀoxide),37,38 thereby the intramolecular
quenching is suppressed, and the chromophore fluorescence is restored. Thus, a judicious choice of the
fluorophore is key to obtain the photophysical and chemical properties that suit the desired analytical
approach. Here, the selected fluorophore has to: (i) exhibit a strong and unambiguous fluorescence
emission (ii) be efficiently quenched by TEMPO radical (iii) be excited at long wavelengths, higher than
350 nm, where DNA does not absorb, and (iv) show little if any absorption in the 260ꢀ330 nm range, in
order to not interfere with the absorbance of the tertꢀbutyl ketone moiety.
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Accordingly, 9ꢀanthraceneacetic acid (AAA) was selected as an appropriate fluorophore because it
fulfills the above requirements; specifically, it has an absorption spectrum that allows selective
excitation at 365 nm and displays a clear structured fluorescence emission spectrum (Figure 1).39
Preliminary steadyꢀstate and timeꢀresolved emission measurements (Figure S2) were performed with
AAA in the presence of 4ꢀhydroxyꢀ2,2,6,6ꢀtetramethylpiperidineꢀ1ꢀoxyl (4ꢀOHꢀTEMPO). In both
experiments, an efficient quenching of the singlet excited state was observed with bimolecular rate
constant of ca. 1010 Mꢀ1 sꢀ1. Altogether, these data support the choice of AAA as the emissive species of
the profluorescent probe.
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Figure 1. Absorption (black) spectrum and emission spectra of AAA (blue) and AAAꢀTEMPO (red) in
acetonitrile, λexc= 365 nm
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Next, synthesis of the new AAAꢀTEMPO dyad was performed following a procedure given in the
literature for similar compounds. In agreement with the expectations, the obtained probe exhibited only
a very weak emission (Figure 1). Then, the ability of AAAꢀTEMPO to react with Cꢀcentered radicals
was assessed by experiments in the presence of 2,2’ꢀazobis(2ꢀamidinopropane) dihydrochloride as
thermolabile radical initiator. An increase of the emission was observed when the azo compound was
heated at 100ºC in the presence of the profluorescent probe (Figure S3). Thus, once established that
AAAꢀTEMPO exhibits the typical behavior of nitroxideꢀbased fluorescent probes, it was used to
investigate the formation of C5 centered radical by irradiation of 1 and 2. When a deaerated acetonitrile
solution of 1 or 2 (0.15 mM) was irradiated at 300 nm in the presence of AAAꢀTEMPO (0.032 mM),
the anthracene emission increased steadily as a function of irradiation time (Figure 2). A control
experiment performed under the same conditions but using a solution of uridine (or thymidine)/AAAꢀ
TEMPO allowed ruling out the occurrence of a Hꢀabstraction process and supported trapping of the C5ꢀ
centered radical. Irradiation of the probe alone in solution revealed its photolability (Figure S4A);
nonetheless, the generated fluorescence was consistently higher in the presence of 1 or 2 (Figure 2).
Steadyꢀstate photolysis was performed in order to confirm the formation of a covalent adduct between
the profluorescent probe and the 5,6ꢀdihydroꢀ2´ꢀdeoxyuridineꢀ or 5,6ꢀdihydrothymidineꢀderived
radicals. Analysis of the samples by UPLCꢀMS confirmed the formation of adducts with an exact mass
m/z 978.5503 and 992.5699, corresponding to the molecular ion [M+H]+ formula C52H84N3O9Si3 and
C53H86N3O9Si3, respectively (Figure S5).
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9 h
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Figure 2. Emission spectra of N2ꢀbubbled acetonitrile solution of AAAꢀTEMPO in the presence of 1
(1:5), irradiated at λ= 300 nm. Inset: I/I0 Ratio as a function of irradiation time for 1 (blue) and 2
(orange); the value obtained for the probe alone has been subtracted
The next step was to characterize the photogenerated C5 radical by laser flash photolysis using 308
nm excitation. Thus, solutions of compounds 1 and 2 (1.24 mM and 2.23 mM, respectively) were
prepared in acetonitrile and degassed under nitrogen atmosphere. The transient absorption spectrum was
registered 4 ꢀs after the laser pulse in order to avoid the presence of misleading shortꢀlived components
such as the triplet excited state. Indeed, this delay was selected because of the long lifetime of the
radicals of interest, which do not decay in a window of tens of microseconds.24,25 Under these
conditions, the transient absorption spectra were noisy, due to the low ε of the radical species; however,
1 exhibited a maximum at ca. 400ꢀ420 nm, whereas in the case of 2 a blue shifted maxima peaking at
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ca. 350ꢀ400 nm were observed (Figure S6). These data are in agreement with previous pulse radiolysis
experiments, thus the obtained transients were assigned to the C5ꢀradicals.23,24
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Table 1. Nature of the electronic transition, CASPT2 vertical absorption energies, oscillator strengths,
and module of the CASSCF dipolar moments relative to the ground state (ꢁꢂ = |ꢀꢁꢂ|ES – |ꢀꢁꢂ|GS) for the
lowꢀlying excited states of the studied species. The experimental absorption band maxima associated
with the corresponding excited state are also shown. Abbreviations: gs = ground state, es = excited state.
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compound
state
nature of the vertical
oscillator strength
(f) gs → es
experimental/eV (nm)
ꢁꢀ/D
transition
absorption
energy/eV(nm)
I
S1
S1
D2
D3
4.11 (302)
0.00004
0.00004
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3.93 (315)
nCO1 → π1∗
nCO1 → π1∗
nCO1 → πSONO
π1 → πSONO
II
Ia
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2.95ꢀ3.10
420)
(400ꢀ
(350ꢀ
D4
D2
D3
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2.74 (453)
3.35 (370)
0.00290
0.00024
0.02372
5.82
π2 → πSONO
IIa
ꢀ1.20
ꢀ3.04
n
CO1→ πSONO
3.54ꢀ3.10
400)
π1 → πSONO
D4
3.82 (325)
0.00235
5.06
π2 → πSONO
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The experimental results were then compared with those obtained by theoretical calculations. For the
sake of simplification, the studies were performed considering the chromophore structures I, II, Ia, and
IIa (Figure 3). The multiconfigurational CASPT2//CASSCF protocol40ꢀ43 was used in combination with
the atomic natural orbital Lꢀtype (ANOꢀL) basis set contracted to C, N, O [4s3p1d]/H [2s1p],44 as
implemented in the MOLCAS 8 suite of programs,45 to compute the vertical absorption energies and the
oscillator strengths of the first nine excited states of the four species (details on the calculations can be
found in the Supporting Information). This computational strategy has been successfully employed in
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•
the theoretical determination of the UVꢀVis spectra of the OH adducts of the pyrimidine DNA/RNA
nucleobases, which have related chemical structures.46
The theoretical results are compiled in Tables 1 and S2, where only the data for the three lowestꢀ
energy excited states are shown. Absorption of UVB light by compounds I and II at low energies
corresponds to the population of the relatively dark S1 state, which mainly involves the nCO1 → π1*
excitation localized in the C=O bond of the pivaloyl fragment (see orbital labeling in Figure S7). Such
excited state was computed vertically at 4.11 eV (302 nm) and 3.93 eV (315 nm) for I and II,
respectively. These theoretical predictions are in reasonable agreement with the experimental band
maxima recorded at 4.35 eV (285 nm) for 1 and 3.93 eV (315 nm) for 2 (see Figure S1). Since the
computed values were obtained for the molecules in vacuo while in the experimental measurements the
compounds were dissolved in acetonitrile, the change of the module of the dipole moments between the
excited and ground states were also analyzed; negligible solvatochromic shifts are expected for the lowꢀ
lying excitedꢀstates of I and II, according to the small ꢁꢀ values (see Tables 1 and S2). Although the
agreement between theory and experiments for the absorption properties of the pivaloyl derivatives of
uracil and thymine was not fully satisfactory, the trend was indeed coincident. Lower absorption
energies were obtained for the compound with the methyl group. A parallel behavior has also been
found when comparing the absorption spectra of the canonical nucleobases uracil and thymine.47
In the case of radical Ia, three excited states (D2, D3, and D4) were computed at the energy range
between 2.5 and 4.0 eV (see Table 1). Taking into account the oscillator strength (f) associated with the
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D1 → D3 transition, which is much higher than that of the D2 and D4 states, the D3 state is predicted as
the main responsible for the broad experimental band peaking at 2.95 – 3.10 eV (400ꢀ420 nm). The
vertical absorption energy of the state, computed at 3.21 eV (386 nm), is in quite good agreement with
the recorded absorption maximum. Regarding the solvent effects, negligible impact on the absorption
energy of the bright D3 state is expected on the basis of its relatively small ꢁꢂ value. The D4 vertical
absorption energy of 3.62 eV (342 nm), the f of the transition, and its estimated red shift, pointed to a
nonꢀnegligible contribution of the D1 → D4 excitation to the broad band. Likewise, the D2 excited state
participates in the ~450 nm region. The f associated with the D1 → D2 transition is much lower than that
of the D1 → D3 one, which is in agreement with the lower absorbance recorded experimentally in this
region. Changes in the localization of the unpaired electron after excitation were tracked by analyzing
the spin densities of the ground and excited states (see Figure 3). The Ia species in the D1 ground state
has the unpaired electron in a π singly occupied natural orbital (SONO) mainly localized at the C5 atom
(see orbitals in Figure S8). The excitation process implies a redistribution of the spin density over the
molecule, changing the radical position to other parts of the molecular structure. Thereby, in the D3
state, the unpaired electron is in the π1 natural orbital (NO) that is now mainly localized over the OCN
atoms, as shown in the spinꢀdensity representations displayed in Figure 3.
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The UVꢀVis absorption properties of the radical IIa are also summarized in Table 1. No relevant
differences in the electronic nature of the ground and excited states were found with respect to those of
Ia. Nevertheless, as a consequence of the methyl group at the C5 position, the vertical absorption
energies are somewhat higher than those of Ia. This blue shift of around 0.1ꢀ0.2 eV is also observed in
the experiments.
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Figure 3. Structures of the models used in the theoretical calculations, I and II are singlet molecules,
whereas Ia and IIa are doublet species. Representation of the difference between the α and β spin
densities obtained from the computed CASSCF wave functions for the ground D1 and excited D3 states
of Ia and IIa using an isovalue of 0.01, red and green colors indicate positive and negative spin
densities, respectively.
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CONCLUSION
In summary, photolytic generation of the lipophilic 5,6ꢀdihydropyrimidinꢀ5ꢀyl radicals in organic
medium has been achieved starting from tertꢀbutyl ketone precursors. These species have been trapped
by TEMPOꢀbased profluorescent probes, and their transient absorption spectra have been obtained by
laser flash photolysis. The experimental data have been corroborated by multiconfigurational ab initio
CASPT2//CASSCF methodology. The obtained precursors should be of interest to investigate the
reactivity of the 5ꢀyl radical in organic solvents, and to check whether 5ꢀhydroperoxyꢀ5,6ꢀ
dihydropyrimidines after reduction to the stable 5ꢀhydroxyꢀ5,6ꢀdihydropyrimidines are detected in the
presence of O2. Furthermore, insertion into defined sequence oligonucleotides could allow studying the
reactivity of peroxyl radicals, once generated, with vicinal bases.
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EXPERIMENTAL SECTION
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Characterization. The H and C NMR spectra were measured by 300 MHz instrument; CDCl3 and
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D2O were used as solvents and the signal corresponding to the solvent taken as the reference: 7.26 ppm
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and 4.79 ppm, respectively for H NMR; and 77.2 ppm for chemical shift of CDCl3 in 13C NMR;
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coupling constants are given in Hz. Exact mass were determined by using a QTof spectrometer coupled
with a liquid chromatography system with a conditioned autosampler at 10ᵒC. The separation was
carried out on an UPLC BEH C18 column (50 mm x 2.1 mm i.d., 1.7 µm). The column temperature was
kept at 40ᵒC. The ESI source was operated in positive ionization mode with the capillary voltage at 1.9
kV. The temperature of the source and desolvation was set at 80°C and 400°C, respectively. The cone
and desolvation gas flows were 20 L h−1 and 800 L h−1, respectively. All data were collected in centroid
mode. Leucineꢀenkephalin was used as the lock mass generating an [M+H]+ ion (m/z 556.2771) at a
concentration of 250 pg/mL and flow rate of 50 ꢂL/min to ensure accuracy during the MS analysis. The
analysis of the irradiation of 1 (or 2) in the presence of AAAꢀTEMPO was achieved under the same
ESI conditions. But, in this case an isocratic elution was performed using 90% acetonitrile and 10%
water (both containing 0.01% formic acid) as the mobile phase during the first 15 minutes followed by a
gradient to reach 100% of acetonitrile at 17 min. The flow was maintained at 0.5 mL/min and the
injection volume was 1ꢂL. The AAAꢀTEMPO probe was characterized by monocrystal ꢀ Ray technique
at 100K temperature.
Photophysical instrumentation. All experiments were performed in a quartz cuvette of 1 cm optical
path. SteadyꢀState fluorescence experiments were performed by means of a spectrofluorometer,
equipped with a Xe lamp (75 W) and a monochromator covering the region of 200ꢀ700 nm. The timeꢀ
resolved fluorescence experiments were carried out with a spectrometer equipped with a pulsed LED
(λexc 340 nm) as excitation source; residual excitation signal was filtered in emission by using a cutꢀoff
filter (50% transmission at 365 nm). For the laser flash photolysis (LFP) experiments, a pulsed excimer
laser instrument with Xe/HCl mixture was used for excitation at 308 nm. The single pulses were of ca.
17 ns duration, and the energy was <100 mJ per pulse. A Xenon lamp was employed as the detecting
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light source. The LFP apparatus consisted of the pulsed laser, the Xe lamp, a monochromator, and a
photomultiplier (PMT) system made up of sideꢀon PMT, housing, and a power supply. The output
signal from the Tektronix oscilloscope was transferred to a personal computer for study. All the
experiments are performed under N2 atmosphere. A system including a Xe lamp (150 W) equipped with
a monochromator was employed for steadyꢀstate monochromatic irradiation. The excitation wavelength
was fixed at 300 nm and the samples were irradiated for 5 h.
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General procedures for synthesis of 1 and 2
2´ꢀDeoxyꢀ5,6ꢀdihydrouridine (1Sa). Synthesis was performed following the method described by
Greenberg et al.,10 0.25 g of the Rh/Al2O3 catalyst were added to 5 g of 2´ꢀdeoxyuridine in 50 mL of
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water under 30 bars of H2. After purification, 4.4 g (96%) of 1Sa was obtained as a colorless oil. H
NMR (300 MHz, D2O) δ 6.26 (t, J = 7Hz, 1H), 4.37 (m, 1H), 3.91 (m, 1H), 3.75 (m, 2H), 3.55 (m, 2H),
2.74 (t, J = 6Hz, 2H), 2.31 (m, 1H), 2.13 (m, 1H). 13C NMR (75 MHz, D2O) δ 173.8 (CO), 154.2 (CO),
85.2 (CH), 84.0 (CH), 70.8 (CH), 61.6 (CH2), 35.7 (CH2), 35.2 (CH2), 30.0 (CH2). HMRS (ESIꢀTOF)
m/z C9H14N2O5Na [M + Na]+ Calcd for 253.0800; Found: 253.0799.
5,6ꢀDihydrothymidine (2Sa). Synthesis was performed following established methods,10,48,49 0.25 g of
the Rh/Al2O3 catalyst were added to 5 g of thymidine in 50 mL of water under 30 bars of H2. After
purification, 4.3 g (85%) of 2Sa was obtained as a colorless oil. 1H NMR (300 MHz, D2O) δ 6.27 (t, J =
6 Hz, 1H), 4.35 (m, 1H), 3.88 (m, 1H), 3.71 (m, 2H), 3.56 (dd, J = 12.9, 5.6 Hz, 1H), 3.25 (m, 1H), 2.83
(m, 1H), 2.30 (m, 1H), 2.14 (m, 1H), 1.21 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, D2O) δ 176.7 (CO),
154.3 (CO), 85.1 (CH), 83.8 (CH), 70.8 (CH), 61.6 (CH2), 42.0 (CH2), 35.4 (CH2), 34.6 (CH), 12.1
(CH3). HMRS (ESIꢀTOF) m/z [M + H]+ Calcd for C10H17N2O5: 245.1137; Found: 245.1131.
2´ꢀDeoxyꢀ3',5'ꢀbisꢀOꢀ[(tertꢀbutyl)dimethylsilyl]ꢀ5,6ꢀdihydrouridine (1Sb). 4.4 g (0.021 mol) of 1Sa,
8.55 g (0.126 mol) of imidazole and 11.4 g (0.076 mol) of tertꢀbutyldimethylsilyl chloride were diluted
in 35 mL of dimethylformamide at 0ᵒC. The mixture was allowed getting to room temperature and
stirred overnight. Then, 250 mL of water were added and extraction was performed with diethyl ether.
The aqueous phase was further washed with diethyl eter (3 x 180 mL). The combined organic phases
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were mixed, washed with brine, dried with MgSO4, and evaporated to dryness under reduced pressure.
Purification was performed by flash column chromatography using hexane/ethyl acetate, 6/1 (v/v) as
eluent, and 7.2 g (76%) of 1Sb was obtained as a white solid. 1H NMR (300 MHz, CDCl3) δ 7.62 (br s,
1H), 6.28 (t , J = 7.0 Hz, 1H), 4.36 (m, 1H), 3.77 (m, 1H), 3.71 (m, 2H), 3.65 (m, 1H), 3.31 (m, 1H),
2.59 (m, 2H), 2.01 (m, 2H), 0.89 (m, 18H), 0.06 (m, 12H). 13C NMR (75 MHz, CDCl3) δ 169.9 (CO),
152.2 (CO), 86.5 (CH), 84.0 (CH), 72.1 (CH), 63.0 (CH2), 37.8 (CH2), 35.4 (CH2), 31.3 (CH2), 26.0
(3CH3), 25.9 (3CH3), 18.4 (C), 18.1 (C), ꢀ4.5 (CH3), ꢀ4.7 (CH3), ꢀ5.3 (CH3), ꢀ5.4 (CH3). HMRS (ESIꢀ
TOF) m/z [M+ Na]+ Calcd for C21H42N2O5Si2Na: 481.2530; Found: 481.2531.
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3',5'ꢀBisꢀOꢀ[(tertꢀbutyl)dimethylsilyl]ꢀ5,6ꢀdihydrothymidine (2Sb). 4.3 g (0.018 mol) of 2Sa, 7.2 g
(0.106 mol) of imidazole and 9.5 g (0.063 mol) of tertꢀbutyldimethylsilyl chloride were diluted in 30
mL of dimethylformamide at 0ᵒC. The mixture was allowed getting to room temperature and stirred
overnight. After addition of 250 mL of water and extraction with diethyl ether, the aqueous phase was
further washed with diethyl ether (3 ×180 mL). The combined organic phases were mixed, washed with
brine, dried with MgSO4, and evaporated under reduced pressure. Purification was performed by flash
column chromatography using hexane/ethyl acetate, 6/1 (v/v) as eluent. Compound 2Sb (6.2 g) was
obtained in 75% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 7.44 (br s, 1H), 6.30 (t, J = 7 Hz,
1H), 4.36 (m, 1H), 3.80 (m, 1H), 3.71 (m, 2H), 3.37 (m, 1H), 3.27 (m, 1H), 2.61 (m, 1H), 1.98 (m, 2H),
1.25 (d, J = 9 Hz, 3H), 0.89 (s + s, 18H), 0.06 (br s, 12H). 13C NMR (75 MHz, CDCl3) δ 172.8 (CO),
152.5 (CO), 86.6 (CH), 83.8 (CH), 72.2 (CH), 63.1 (CH2), 42.1 (CH2), 38.1 (CH2), 35.6 (CH), 26.0
(3CH3), 25.9 (3CH3), 18.5 (C), 18.1 (C), 13.2 (CH3), ꢀ4.5 (CH3), ꢀ4.7 (CH3), ꢀ5.3 (CH3), ꢀ5.4 (CH3).
HRMS (ESIꢀTOF) m/z [M+ H]+ Calcd for C22H45N2O5Si2: 473.2867; Found: 473.2862.
2´ꢀDeoxyꢀ3',5'ꢀbisꢀOꢀ[(tertꢀbutyl)dimethylsilyl]ꢀ3ꢀ[[2ꢀ(trimethylsilyl)ethoxy]methyl]ꢀ5,6ꢀdihydrouridine
(1Sc). A solution of 1Sb (7.23g, 0.016 mol) in 11 mL of N,Nꢀdiisopropylethylamine was mixed with 4.2
mL (0.024 mol) of 2ꢀ(trimethylsilyl)ethoxymethyl chloride diluted in 10 mL of CH2Cl2, and the mixture
was stirred overnight at room temperature. Then, CH2Cl2 and a saturated solution of NaHCO3 were
added and the organic phase was extracted, dried with MgSO4 and concentrated under reduced pressure.
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Purification was performed by flash column chromatography using 10:1 hexane/ethyl acetate (v/v) as
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eluent, and 5.3 g (57%) of 1Sc was obtained as a colorless oil. H NMR (300 MHz, CDCl3) δ 6.32 (t, J
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= 7Hz, 1H), 5.21 (m, 2H), 4.34 (m, 1H), 3.83ꢀ3.56 (m,6H), 3.25 (m, 1H), 2.68 (m, 2H), 1.99 (m, 2H),
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0.95ꢀ0.88 (m, 20H), 0.05 (brs , 12H), ꢀ0.02 (brs, 9H). C NMR (75 MHz, CDCl3) δ 169.7(CO), 152.9
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(CO), 86.4 (CH), 84.8(CH), 72.0 (CH), 69.9 (CH2), 67.0 (CH2), 63.0 (CH2), 37.9 (CH2), 34.3 (CH2),
32.0 (CH2), 25.9 (3CH3), 25.8 (3CH3), 18.4 (CH2), 18.2 (2C), ꢀ1.3 (3CH3), ꢀ4.5 (CH3), ꢀ4.7 (CH3), ꢀ5.4
(CH3), ꢀ5.5 (CH3). HMRS (ESIꢀTOF) m/z [M+ Na]+ Calcd for C27H56N2O6Si3Na: 611.3344; Found:
611.3326.
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3',5'ꢀBisꢀOꢀ[(tertꢀbutyl)dimethylsilyl]ꢀ3ꢀ[[2ꢀ(trimethylsilyl)ethoxy]methyl]ꢀ5,6ꢀdihydrothymidine (2Sc).
A solution of 2Sb (4.3 g, 0.0091 mol) in 16 mL of N,Nꢀdiisopropylethylamine was mixed with 3.5 mL
(0.02 mol) of 2ꢀ(trimethylsilyl)ethoxymethyl chloride diluted in 10 mL of CH2Cl2, and the mixture was
stirred overnight at room temperature. Then, CH2Cl2 and a saturated solution of NaHCO3 were added
and the organic phase was extracted, dried with MgSO4 and concentrated under reduced pressure.
Purification was performed by flash column chromatography using 10:1 hexane/ethyl acetate (v/v) as
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eluent, and 4.2 g (53%) of 2Sc was obtained as colorless oil. H NMR (300 MHz, CDCl3) δ 6.35 (t, J =
9Hz, 1H), 5.20 (s, 2H), 4.35 (m, 1H), 3.79 (m, 1H), 3.70 (m, 2H), 3.61 (m, 2H), 3.29 (m, 2H), 2.66 (m,
13
1H), 1.95 (m, 2H), 1.25 (d, J = 9Hz, 3H), 0.90 (m, 20H), 0.06 (brs, 12H), ꢀ0.01 (s, 9H). C NMR (75
MHz, CDCl3) δ 172.8 (CO), 153.1 (CO), 86.5 (CH), 84.5 (CH), 72.2 (CH), 69.9 (CH2), 66.9 (CH2), 63.1
(CH2), 40.8 (CH2), 38.1 (CH2), 36.0 (CH), 26.0 (3CH3), 25.9 (3CH3), 18.5 (2C), 18.2 (CH2), 13.6 (CH3),
ꢀ1.3 (3CH3), ꢀ4.5 (CH3), ꢀ4.7 (CH3), ꢀ5.3 (CH3), ꢀ5.4 (CH3). HMRS (ESIꢀTOF) m/z [M + Na]+ Calcd for
C28H58N2O6Si3Na: 625.3500; Found: 625.3484.
2´ꢀDeoxyꢀ3',5'ꢀbisꢀOꢀ[(tertꢀbutyl)dimethylsilyl]ꢀ3ꢀ[[2ꢀ(trimethylsilyl)ethoxy]methyl]ꢀ5ꢀ(2,2ꢀdimethylꢀ1ꢀ
oxopropyl)ꢀ5,6ꢀdihydrouridine (1). The final compound 1 was obtained as a diastereomeric mixture
following the methodology described in the literature by Greenberg and col. for related compounds.27
Briefly, a solution of 1Sc (0.1 g, 1.7 10ꢀ4 mol) in tetrahydrofuran was stirred during 30 min with 0.85
mL of 2M LDA at 78ᵒC. Then, pivaloyl chloride (0.02 mL, 1.6 10ꢀ4 mol) was added dropwise and the
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mixture was kept at this temperature for 30 min. The organic phase was extracted after addition of a
saturated solution of NH4Cl and ethyl acetate (3 × 75 mL), washed with brine, dried with MgSO4 and
evaporated to dryness under reduced pressure. The purification was performed by preparative liquid
chromatography (PLC) using 5:1 hexane/acetate (v/v) as eluent to afford compound 1 as yellowish oil
(0.06 g, 53%). 1H NMR (300 MHz, CDCl3) δ 6.34 (dd, J = 9Hz, J = 6Hz, 1H), 5.28 (d, J = 9Hz, 1H),
5.15 (d, J = 9Hz, 1H), 4.31 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.67ꢀ3.57 (m, 5H), 3.47 (m, 1H), 1.93
(m, 2H), 1.25ꢀ1.21 (m, 9H), 0.89 (m, 20H), 0.07 ꢀ ꢀ0.01 (m, 21H).13C NMR (75 MHz, CDCl3) δ 209.1
(CO), 207.6 (CO), 168.1 (CO), 167.9 (CO), 152.7 (CO), 152.2 (CO), 86.8 (CH), 86.7 (CH), 85.1 (CH),
84.8 (CH), 77.4 (C), 72.7 (CH), 72.4 (CH), 70.0 (CH2), 67.1 (CH2), 67.0 (CH2), 63.6 (CH2), 63.3 (CH2),
47.9 (CH), 47.7 (CH), 45.5 (C), 38.2 (CH2), 37.6 (CH2), 37.4 (CH2), 26.0 (3CH3), 25.9 (3CH3), 25.7
(3CH3), 18.4 (C), 18.1 (CH2), ꢀ1.3 (3CH3), ꢀ4.5 (CH3), ꢀ4.7 (CH3), ꢀ5.3 (CH3), ꢀ5.4 (CH3). HRMS (ESIꢀ
TOF) m/z [M + Na]+ Calcd for C32H64N2O7Si3Na: 695.3919; Found: 695.3903.
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3',5'ꢀBisꢀOꢀ[(tertꢀbutyl)dimethylsilyl]ꢀ3ꢀ[[2ꢀ(trimethylsilyl)ethoxy]methyl]ꢀ5ꢀ(2,2ꢀdimethylꢀ1ꢀoxopropyl)
ꢀ5,6ꢀdihydrothymidine (2). The final compound 2 was obtained following the methodology described in
the literature by Greenberg and col. for related compounds.27 Briefly, a solution of 2Sc (0.1 g, 1.7 10ꢀ4
mol) in tetrahydrofuran was stirred during 3h with 0.83 mL of 2 M LDA at 78ᵒC. Then, pivaloyl
chloride (0.02 mL, 1.6 10ꢀ4 mol) was added dropwise and the mixture was kept at this temperature for
1h 30 min, allowed to reach room temperature and stirred for additional 3h. The organic phase was
extracted after addition of a saturated solution of NaHCO3 and ethyl acetate (3 × 75 mL), washed with
brine, dried with MgSO4 and evaporated to dryness under reduced pressure. The purification was
performed by preparative liquid chromatography (PLC) using 5:1 hexane/acetate (v/v) as eluent to
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afford compound 2 as transparent oil (0.07 g, 61%). H NMR (300 MHz, CDCl3) δ 6.39 (t, J = 6 Hz,
1H), 4.93 (m, 2H), 4.33 (m, 1H), 3.94 (d, J = 12Hz, 1H), 3.75 ꢀ 3.63 (m, 4H), 3.50 (t, J = 6Hz, 2H), 2.09
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(m, 1H), 1.89 (m, 1H), 1.49 (s, 3H), 1.30 (s, 9H), 0.89 (m, 20H), 0.06 (br s, 12H), ꢀ0.02 (s, 9H). C
NMR (75 MHz, CDCl3) It corresponds to the enol ester 2´ resulting from 1,3 acyl shift inside the NMR
tube. δ 174.6 (CO), 153.8 (CO), 134.3 (CO), 94.1 (C), 86.0 (CH), 84.4 (CH), 72.1 (CH), 71.2 (CH2),
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65.0 (CH2), 63.0 (CH2), 42.1 (CH2), 39.2 (C), 36.2 (CH2), 27.1 (3CH3), 26.1 (3CH3), 25.9 (3CH3), 18.5
(C), 18.1 (C), 18.0 (CH2), 12.8 (CH3), ꢀ1.2 (3CH3), ꢀ4.5 (CH3), ꢀ4.7 (CH3), ꢀ5.2 (CH3), ꢀ5.4 (CH3).
HMRS (ESIꢀTOF) m/z [M + H]+ Calcd for C33H67N2O7Si3: 687.4256; Found: 687.4271.
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2,2,6,6ꢀTetramethylꢀ4ꢀ(9ꢀanthracenylacetoxy)ꢀpiperidineꢀ1ꢀoxyl (AAA-TEMPO). The profluorescent
probe AAAꢀTEMPO was obtained by esterification of 2ꢀ(anthracenꢀ9ꢀyl)acetic acid with 4ꢀhydroxyꢀ
TEMPO, adapting a methodology described by Scaiano et al.50 Briefly, 2ꢀ(anthracenꢀ9ꢀyl)acetic acid
(0.2 g, 8.5 10ꢀ4 mol)51, dicyclohexylcarbodiimide (0.2 g, 9.3 10ꢀ4 mol), 4ꢀhydroxyꢀTEMPO (0.16 g, 9.3
10ꢀ4 mol) and 4ꢀDMAP (0.01 g, 8.5 10ꢀ5 mol) were diluted with 4 mL of CH2Cl2 at room temperature
and stirred until the esterification was completed (6 h). The precipitated N,Nꢀdicyclohexyl urea was
filtered off, and the filtrate was washed with H2O (3 x 30 mL), 5% acetic acid solution (3 x 5 mL), and
once more with H2O (3 x 50 mL), dried with MgSO4 and evaporated to dryness. Purification was
performed by flash column chromatography using hexane/ethyl acetate 4:1 (v/v) as eluent to afford
AAAꢀTEMPO as a coralꢀcolored crystalline solid (0.256 g, 75%). The presence of the nitroxide
prevented the characterization by NMR spectroscopy, thus this compound was characterized by XꢀRay
(CCDC 1444537, See Supporting Information).
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Experimental details of fluorescence studies.
Intermolecular quenching of AAA by 4-OH-TEMPO. A solution of 2ꢀ(anthracenꢀ9ꢀyl) acetic acid AAA
in acetonitrile (2.6 × 10ꢀ5 M) was prepared in a cuvette. On the other hand, a stock solution of 4ꢀOHꢀ
TEMPO (0.1 M) was prepared, so it was only necessary to add microliter volumes to the sample cell to
obtain appropriate concentration of the quencher. Quenching experiments were performed using an
excitation wavelength λexc of 365 and 375 nm for steadyꢀstate and timeꢀresolved studies, respectively.
The rate constants for the reaction of AAA with 4ꢀOHꢀTEMPO were obtained from the SternꢀVolmer
plots52 following equation (1) or (2) for steadyꢀstate and timeꢀresolved measurements, respectively.
I0/I= 1 + KSV × [4ꢀOHꢀTEMPO] = 1 + kq × τ0 × [4ꢀOHꢀTEMPO] (1)
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where I0 is the fluorescence intensity of AAA in the absence of quencher, I the emission intensity after
addition of a quencher concentration [4ꢀOHꢀTEMPO] in molar and KSV the SternꢀVolmer constant (Mꢀ
1). A value of 109.4 Mꢀ1 was obtained for KSV (Figure S2A).
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1/τ = 1/τ0 + kq x [4ꢀOHꢀTEMPO]
(2)
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where τ is the lifetime after addition of a quencher concentration [4ꢀOHꢀTEMPO] and τ0 the lifetime for
AAA in the absence of quencher. A value τ0 of 4.7 ns was measured (Figure S2B), and a bimolecular
quenching rate constant kq of ca. 2 x 1010 Mꢀ1 sꢀ1 was obtained from the SternꢀVolmer plot (Figure S2B,
inset). Moreover, it appears from equation (1) that kq can be determined from KSV. A value kq of 2.3
1010 Mꢀ1sꢀ1 was thus obtained for the steadyꢀstate experiment, which rules out the occurrence of static
quenching.
Reactivity of AAA-TEMPO with 2,2´ꢀazobis(2ꢀamidinopropane) dihydrochloride. An acetonitrile
solution containing AAAꢀTEMPO (0.03 mM) and the radical initiator 2,2’ꢀazobis(2ꢀamidinopropane)
dihydrochloride (0.03mM) was heated at 100ᵒC during 8 h, and spectra were registered at different
incubation times using an excitation wavelength of 365 nm (Figure S3).
Reactivity of AAA-TEMPO with 1 and 2. Solutions containing AAAꢀTEMPO (0.03 mM) and 1 or 2
(0.15 mM) were irradiated monochromatically at 300 nm up to 9 h. Emission spectra were registered at
different irradiation times by exciting at 365 nm. A solution containing the probe alone was also
monitored as control experiment (Figure S4).
Experimental details of monochromatic irradiation of AAAꢀTEMPO in the presence of 1 or 2.
Solutions containing AAAꢀTEMPO in the presence of 1 or 2 in a 1:5 ratio, were irradiated
monochromatically at 300 nm for 5 h. The solutions were concentrated and analyzed by UPLCꢀHRMS
(Figure S5).
Computational details. The theoretical spectra of the species I, II, Ia, and IIa have been computed by
means of the completeꢀactiveꢀspace secondꢀorder perturbation theory (CASPT2//CASSCF) protocol.
This means that the groundꢀstate geometries have been optimized at the CASSCF level and then the
vertical excitation energies have been calculated with the CASPT2 method at the CASSCF optimized
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geometry. The atomic natural orbital Lꢀtype (ANOꢀL) contracted to C, N, O [4s3p1d]/H [2s1p] basis set
has been used in all the calculations.
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For the groundꢀstate geometry optimizations, the multiconfigurational CASSCF wave functions have
been built by including only the most relevant π molecular orbitals (MOs) in the active space. The
following criterion has been used to choose the active orbitals: orbitals with occupations numbers in the
groundꢀstate CASSCF calculations higher than 1.98 and lower than 0.02 are kept inactive, while natural
orbitals with values in the range 1.98ꢀ0.02 are placed in the CAS space. The total number of active
electrons and active MOs for each compound is summarized in Table S1. Thus, three π bonding MOs
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localized in the C=O bonds and the related π* antibonding MOs have been included in the CAS for
compounds I and II. On the other hand, two π MOs localized in the C=O bonds, the singlyꢀoccupied
natural orbital (SONO) mainly centered at C5 position, and the two corresponding C=O π* MO have
formed the active space of the Ia and IIa species. Only one root has been demanded in the CASSCF
procedure for the groundꢀstate optimizations.
For the vertical absorption energies, stateꢀaverage (SA)ꢀCASSCF calculations have been performed
using 10 roots. All valence MOs have been included in the active space (see Table S1 and Figures S7
and S8). Calibration of the basis set, active space, and number of roots have been documented
elsewhere.46 All valence π and π* MOs plus the lone pair orbitals of the oxygen atoms (nCO) have been
included in the active space (see Table S1 and Figures S7 and S8). The occupation numbers of the SAꢀ
CASSCF computations were used for this selection using the criterion described above. The dynamical
electron correlation energies have been computed by means of perturbation theory with the CASPT2
method, using the SAꢀCASSCF wave function as reference. The zerothꢀorder Hamiltonian has been
employed as originally implemented [ionization potential electron affinity (IPEA) parameter with a
value of 0.0 au]. To minimize the effect of weak intruder states, the parameter of the imaginary level
shift53 has been fixed to 0.2 au.
Oscillator strengths (ꢃ) have been computed according to the formula
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2
ꢃ = EꢄꢅTDMꢆ
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where Eꢄꢅ refers to the CASPT2 vertical transition energy. The TDM stands for the transition dipole
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ꢁꢂ
ꢁꢂ
moment between the initial φꢇ and final φꢆ CASSCF wave functions, TDM = ꢈφꢇꢉdꢉφꢆꢊ, where d is
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the dipole moment operator.
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All the calculations have been conducted with the MOLCAS 8 software package.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
NMR spectra, XꢀRay, absorption and emission spectra, and computational details (PDF)
Xꢀray data for AAAꢀTEMPO (CIF)
AUTHOR INFORMATION
Corresponding Author
lvirgini@itq.upv.es, mmiranda@qim.upv.es
ACKNOWLEDGMENT
Spanish Government (CTQ2012ꢀ32621, CTQ2015ꢀ70164ꢀP, CTQ2014ꢀ58624ꢀP, RIRAAF RETICS
RD12/0013/0009, Severo Ochoa program/SEVꢀ2012ꢀ0267, María de Maetzu program/MDMꢀ2015ꢀ
0538, BESꢀ2011ꢀ048326 and BESꢀ2013ꢀ066566, JCIꢀ2012ꢀ13431) and Generalitat Valenciana
(Prometeo II/2013/005 and GV2015ꢀ057) are gratefully acknowledged. Dr M. Luisa Marin is also
acknowledged for her help during laser flash photolysis experiments.
REFERENCES
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