Independent Generation and Reactivity of 2′-Deoxyguanosin- N1-yl Radical

Article abstract of DOI:10.1021/acs.joc.0c01095

2′-Deoxyguanosin-N1-yl radical (dG(N1-H)?) is the thermodynamically favored one-electron oxidation product of 2′-deoxyguanosine (dG), the most readily oxidized native nucleoside. dG(N1-H)? is produced by the formal dehydration of a hydroxyl radical adduct of dG as well as by deprotonation of the corresponding radical cation. dG(N1-H)? were formed as a result of the indirect and direct effects of ionizing radiation, among other DNA damaging agents. dG(N1-H)? was generated photochemically (λmax = 350 nm) from an N-aryloxy-naphthalimide precursor (3). The quantum yield for photochemical conversion of 3 is ～0.03 and decreases significantly in the presence O2, suggesting that bond scission occurs from a triplet excited state. dG is formed quantitatively in the presence of excess β-mercaptoethanol. In the absence of a reducing agent, dG(N1-H)? oxidizes 3, decreasing the dG yield to ～50%. Addition of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) as a sacrificial reductant results in a quantitative yield of dG and two-electron oxidation products of 8-oxodGuo. N-Aryloxy-naphthalimide 3 is an efficient and high-yielding photochemical precursor of dG(N1-H)? that will facilitate mechanistic studies on the reactivity of this important reactive intermediate involved in DNA damage.

Full text of DOI:10.1021/acs.joc.0c01095

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Independent Generation and Reactivity of 2′-Deoxyguanosin‑N1‑yl
Radical
Liwei Zheng and Marc M. Greenberg*
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ABSTRACT: 2′-Deoxyguanosin-N1-yl radical (dG(N1-H)•) is the
thermodynamically favored one-electron oxidation product of 2′-
deoxyguanosine (dG), the most readily oxidized native nucleoside.
dG(N1-H)• is produced by the formal dehydration of a hydroxyl
radical adduct of dG as well as by deprotonation of the
corresponding radical cation. dG(N1-H)• were formed as a result
of the indirect and direct effects of ionizing radiation, among other
DNA damaging agents. dG(N1-H)• was generated photochemically
(λmax = 350 nm) from an N-aryloxy-naphthalimide precursor (3).
The quantum yield for photochemical conversion of 3 is ∼0.03 and decreases significantly in the presence O2, suggesting that bond
scission occurs from a triplet excited state. dG is formed quantitatively in the presence of excess β-mercaptoethanol. In the absence
of a reducing agent, dG(N1-H)• oxidizes 3, decreasing the dG yield to ∼50%. Addition of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-
oxodGuo) as a sacrificial reductant results in a quantitative yield of dG and two-electron oxidation products of 8-oxodGuo. N-
Aryloxy-naphthalimide 3 is an efficient and high-yielding photochemical precursor of dG(N1-H)• that will facilitate mechanistic
studies on the reactivity of this important reactive intermediate involved in DNA damage.
INTRODUCTION
■
2′-Deoxyguanosine (dG) is the most readily oxidized native
nucleoside.1,2 The radical cation (dG•+) resulting directly from
one-electron oxidation is critically important for hole transfer in
DNA.3,4 In contrast, deprotonation of dG•+ from the N2-amino
group or the N1-position to produce dG(N2−H)• and dG(N1−
H)•, respectively, squelches electron transfer (Scheme 1).5,6
dG(N2-H)• and dG(N1-H)• are also produced via formal
dehydration of the C4-hydroxyl radical adduct of dG via an
ion pair (C4−OH).7 Of the two neutral radicals, dG(N1-H)• is
accepted to be lower in energy.8 It is notable that dG(N1-H)•
is produced by both major pathways of γ-radiolysis, the
“direct” and “indirect” effects.9 Despite the importance
of dG(N1-H)• in DNA oxidation chemistry, there is
limited understanding of its reactivity. dG(N1-H)• is a
component of mixtures produced from (pulse) radiolysis of
dG.10 Previous studies revealed that it is unreactive with
O2.11,12 Photolabile nucleosides are useful tools for generating
and studying reactive intermediates involved in nucleic acid
oxidation.13−17 Precursors 1 and 2 were designed to
independently generate dG(N1-H)•, but mass balances were
38%.18,19 We wish to report on a photochemical precursor for
dG(N1-H)• that provides the radical in high yield based upon
mass balances in photolysates.
Although dG(N1-H)• is lower in energy than dG(N2-H)•,
the difference in the barriers to their formation from the C4−
OH nucleoside is calculated to be <1 kcal/mol.7 The
relationship between dG(N2-H)• and dG(N1-H)• is more
complicated in duplex DNA in which the former is the ultimate
deprotonation product of dG•+ due to base pairing with dC.6
In addition, contrary to reports that hydroxyl radical
preferentially adds to the purine π-bonds, direct formation of
dG(N2-H)• by hydrogen atom abstraction has been proposed
to be the major pathway for the reaction between dG and this
species.10,20−24 dG(N2-H)• is then proposed to isomerize to
dG(N1-H)• on the submillisecond time scale. High-yielding
methods independently generating dG(N2-H)• and dG(N1-
Received: May 5, 2020
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Scheme 1. Formation of dG(N1-H)· and dG(N2-H)
Scheme 2. Generation of dG(N1-H)• from the Photolysis of 3
a
Scheme 3. Synthesis of dG(N1-H)• Precursor 3
a
Key (a) (i) DABCO, DMF; (ii) 4, diazabicycloundecene (DBU). (b) Et3N·3HF, tetrahydrofuran (THF).
H)• are desirable to resolve the uncertainties and complexity of
their reactivities. dG(N2-H)• and dG(N1-H)• have been
produced from photochemical precursors, but the methods
are inefficient and/or do not provide suitable mass
balances.16,18,19
produced from purine oxidation are more limited.15,16,25 The
N-hydroxypyrid-2(1H)-one (1) and the corresponding thione
(2) were designed to produce dG(N1-H)• upon UV
photolysis.18,19 The former proved to be a photochemical
source of dG(N1-H)•, but the mass balance was <40%. All of
the published precursors to heteroatom-centered purine
radicals utilize cleavage of weak N−N or N−O bonds.25
Similarly, we pursued N-aryloxy-1,8-naphthalimide 3 (Scheme
2). Other N-oxy-1,8-naphthalimides undergo efficient N−O
bond cleavage under UV excitation.26,27 Furthermore, we
postulated that 3 would exhibit strong absorption at wave-
RESULTS AND DISCUSSION
■
Design and Preparation of a dG(N1-H)• Precursor.
Independent generation of nucleoside radicals and the
analogous nucleotide radicals in oligonucleotides has greatly
improved our understanding of nucleic acid oxidation.14
However, examples of studies on the heteroatom radicals
B
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Figure 1. HPLC chromatograms (260 nm detection) of the photolyses of 3 (0.1 mM) under anaerobic conditions in a phosphate buffer (10 mM,
pH = 7.2). (a) Photolyses of 3 in the absence of BME; (b) photolysis of 3 in the presence of BME (10 mM). The peak at 11 min is attributed to a
byproduct generated from BME.
lengths >300 nm, a desirable property when working with
nucleic acids.
naphthalimide (NP, Scheme 2). While the contributions of
homocleavage and photoreduction are uncertain, the quanti-
tative generation of dG suggests that dG(N1-H)• is reduced by
BME. These observations are in contrast to a previous study, in
which dG(N1-H)• was generated from 1 and was reported to
not react with BME.19 Suitable reducing agents, albeit not
BME, gave rise to similar increases in yields of dA and mass
balances from photolyses of 7.15 The product at 16 min was
identified as NP by ultraperformance liquid chromatography-
mass spectrometry (UPLC-MS) and high-performance liquid
chromatography (HPLC) (Figure 2). The lack of formation of
Precursor 3 was readily synthesized using the method
developed by Lakshman, in which N-hydroxy-1,8-naphthali-
mide (4) was reacted with 5 that was activated by 1,4-
diazabicyclo[2.2.2]octane (DABCO) (Scheme 3).28 Substitu-
tion of the sulfonate in 5 was straightforward when
dimethylformamide (DMF) was substituted for the more
commonly used DME solvent, due to poor solubility of 4 in
the latter. Upon removing the tert-butyldimethylsilyl
(TBDMS) protecting groups from 6, precursor 3 was obtained.
The UV spectrum of 3 shows absorption maxima at 287 nm (ε
= 1.1 × 104 M−1 cm−1), which arises from the guanine, and
another at 346 nm (ε = 1.72 × 104 M−1 cm−1), which are
attributed to the guanine and 1,8-naphthalimide moieties,
respectively (Figure S4).
Photochemical Generation of dG(N1-H)• from 3. When
photolyzed in the absence of O2, 3 (0.1 mM) is completely
consumed in less than 7 min. The quantum yield of the
disappearance of 3 (Φ = 0.03) was determined using N-
hydroxy-1,8-naphthalimide triflate as an actinometer26,27 and is
20 times higher than that of the precursor (7) for 2′-
deoxyadenosin-N6-yl radical under the same photolysis
conditions.15 This increased quantum yield and the high
absorbance around 350 nm contribute to the rapid conversion
of 3 (Figure S4).
Figure 2. HPLC chromatograms (260 nm detection) of photolysates
(5 min) of 3 (0.1 mM) under anaerobic conditions in a phosphate
buffer (10 mM, pH = 7.2) (a) in the presence of BME (10 mM); (b)
in the absence of BME. (c) Solution containing dT (0.1 mM) and NP
(0.1 mM). The complete chromatogram is present in Figure S5.
N-hydroxy-1,8-naphthalimide (4) was consistent with homo-
lytic N−O bond cleavage and excludes the generation of dG
via hydrolysis. The yield of 1,8-naphthalimide (NP) was
quantitative and, unlike that of dG, was independent of the
presence of BME. This is an important observation for
interpreting the photochemistry of 3 (vide infra).
dG was detected (52
2%) in the absence of β-
mercaptoethanol (BME, Figure 1a). As in the photochemical
studies of 7, the generation of dG in the absence of reducing
agents may be attributed to reduction of dG(N1-H)• by the
radical precursor (3). In contrast, dG was formed in nearly
quantitative yield when the photolysis was carried out in the
presence of 10 mM thiol (Figure 1b). Furthermore, the
consumption rate of the precursor was greater in the presence
of thiol. We propose that the accelerated photoconversion is
caused by the photoreduction of 3 by BME, and the ensuing
fragmentation leads to the formation of dG(N1-H)• and 1,8-
The consumption rate of 3 decreased significantly when the
photolyses were carried out in the presence of O2 (Figure 3).
The effect of oxygen on the consumption rate of 3 is consistent
with the report that the 1,8-naphthalimide triplet excited state
29
1
is efficiently quenched by O2 to yield O2 (Scheme 2). This
observation indicates that the triplet excited state of 3
undergoes homolytic bond scission to release dG(N1-H)•.
Photophysical studies on 1,8-naphthalimide photoacid gen-
erators indicate that bond scission occurs from the singlet
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Figure 3. HPLC chromatograms (260 nm detection) of the photolyses of 3 (0.1 mM) under aerobic conditions in a phosphate buffer (10 mM, pH
= 7.2). (a) Photolyses of 3 in the absence of BME; (b) photolysis of 3 in the presence of BME (10 mM). The peak at 11 min is attributed to a
byproduct generated from BME.
Scheme 4. Photolysis Product from 3 in the Absence of Exogenous Reductant
excited state.27 Although we cannot rule out a contribution of
the singlet excited state to the formation of dG(N1-H)•, the
involvement of the triplet excited state in this chemistry is
different from other 1,8-naphthalimides. In the absence of
photolysis in the presence of O2 eventually led to the reduction
of the yield of dG.
Secondary Redox Reactions in the Absence of
Exogenous Reducing Agent (BME). To explain the
formation of dG at ∼50% yield in the absence of BME, we
propose that precursor (3) serves as the reducing agent of
dG(N1-H)• but the fate of the portion of the sacrificed
precursor was unclear. As previously mentioned, the yield of
NP was quantitative under anaerobic conditions regardless of
the presence of BME. This observation provided us a hint of
the possible products generated from the oxidation of
precursor 3 by dG(N1-H)• and naphthalimidyl radical
(NP•). Scheme 4 shows a formal representation of the
photolysis, in which two products detected by HPLC, dG, and
NP, result from the single electron reduction by 3. This
behavior is consistent with previous reports on the reactivity of
purine radicals and similar types of precursors as employed
here.15,16 To balance the chemical equation electronically, we
postulate that the 50% of 3 that acts as a reducing agent is
ultimately converted into three-electron oxidized forms of
dG(N1-H)• (four-electron oxidized forms of dG), such as dSp
and dGh (Scheme 4).35 This proposal was corroborated by
utilizing Burrow’s previous report on the separation and
identification of dSp and dGh.35 As previously reported, the
photolysates were initially analyzed by C18-reversed phase
thiol, 81
1% of the precursor was consumed in 3 h (as
opposed to <7 min under anaerobic conditions, Figure 1a),
and the yield of dG was only 14 1%. We propose that the
1
low yield of dG was due to oxidation by O2, produced from
quenching of triplet excited states of 3 and NP by
oxygen.22,30−33 This proposal was supported by the observa-
tion that the yield of dG based on conversion decreased as the
photolysis time increased. The yield of dG based on
unrecovered 3 decreased from 57 1% at 1 h to 37 4%
at 2 h (Figure 3a), indicating that continued photolysis of the
mixture of dG and NP in the presence of O2 led to destruction
of the former. Including BME (10 mM) in aerobic photolyses
resulted in a significantly different outcome. dG was formed
quantitatively, and 3 was completely consumed under these
conditions within 1 h (Figure 3b). The accelerated
consumption of the precursor is similar to that observed
under anaerobic conditions, indicating a possible photo-
reduction pathway. In addition, BME consumes O2 by reacting
with singlet O2, which may contribute to the increased rate of
photoconversion as well as higher dG yield.34 Extended
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Figure 4. UPLC-MS/MS analysis of the anaerobic photolysis of 3. (a) Extracted-ion chromatogram (EIC) of dGh (calculated (M + H) m/z =
274.1146, observed m/z = 274.1143) and (b) EIC of dSp and Sp (calculated (M + H) m/z = 184.0465, observed m/z = 184.0468).
Scheme 5. Proposed Mechanism for Secondary Redox Chemistry
HPLC, and the void volume containing the polar four-electron
oxidized guanine 2′-deoxynucleosides was collected. The void
volume was then analyzed by UPLC-MS equipped with a
Hypercarb column, which revealed dSp and dGh (Figure 4).
Our proposed mechanism (Scheme 5) begins with UV-
excited 3 undergoing homolytic cleavage of the N−O bond to
generate dG(N1-H)• and NP• (Scheme 5a). Precursor 3
reduces dG(N1-H)• or NP• to generate the radical cation 9
and dG or NP, respectively (Scheme 5b, oxidation of 3 by NP•
is not shown). Fragmentation of 9 upon reaction with water
and subsequent tautomerization results in the formation of 8-
oxodGuo and NP• (Scheme 5b). Given the relatively low
oxidation potential of 8-oxodGuo (0.74 V/normal hydrogen
electrode (NHE)), both dG(N1-H)• (1.29 V/NHE) and NP•
(>2 V/NHE) can be reduced by it, and 8-oxodGuo undergoes
two-electron oxidation to generate products such as dGh and
dSp (Scheme 5c, oxidation of 8-oxodGuo by dG(N1-H)• is not
shown).1,19,36−40 It is also possible that 3 in the excited state
oxidizes ground state 3 and 8-oxodGuo, which would lead to
the same result. To demonstrate whether 8-oxodGuo can serve
as the reducing agent, 3 (0.1 mM) was photolyzed in the
presence of two equivalents of 8-oxodGuo (Figure 5), a much
lower concentration than that of BME (10 mM) used (Figure
1b). The addition of 8-oxodGuo restored the quantitative
formation of dG observed under anaerobic conditions in the
presence of BME, and one equivalent of 8-oxodGuo was
consumed. The yields of dSp and dGh were qualitatively
higher for the photolyses carried out in the presence of 8-oxo-
dG as expected (Figure 6).
CONCLUSIONS
■
We developed a precursor (3) that generates the thermody-
namically favored neutral purine radical of 2′-deoxyguanosine
(dG(N1-H)•) in high yield. The efficiency of radical formation
is also good, particularly under anaerobic conditions. High
yields of dG are obtained in the presence of a reducing agent,
β-mercaptoethanol. In the absence of exogenous reducing
agent, 3 serves as a reducing agent, ultimately producing the
four-electron dG lesions, dSp, and dGh. Dioxygen effects on
the photochemistry of 3 indicate that contrary to other 1,8-
naphthalimides, cleavage to produce dG(N1-H)• occurs from
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flash chromatography on a silica column. Elution with 50% EtOAc in
hexanes gave 6 as a yellow solid (64 mg, 46%). 1H NMR (400 MHz,
CDCl3) δ 8.63 (d, J = 7.3 Hz, 2H), 8.27 (d, J = 8.2 Hz, 2H), 7.95 (s,
1H), 7.78 (t, J = 7.8 Hz, 2H), 6.26 (t, J = 6.2 Hz, 1H), 4.55 (dd, J =
9.2, 4.3 Hz, 1H), 3.99−3.89 (m, 1H), 3.80 (dd, J = 11.2, 3.0 Hz, 1H),
3.73 (dd, J = 11.2, 3.0 Hz, 1H), 2.50 (dt, J = 12.4, 6.1 Hz, 1H), 2.41−
2.25 (m, 1H), 0.89 (s, 9H), 0.85 (s, 9H), 0.08 (s, 6H), 0.04 (s,3H),
and 0.03 (s, 3H). 13C {1H} NMR (101 MHz, CDCl3) δ 159.9, 158.7
155.18, 139.2, 134.7, 131.9, 131.8, 127.7, 127.0, 122.7, 113.8, 87.5,
83.8, 71.4, 62.5, 41.1, 25.9, 25.7, 18.3, 17.9, −4.6, −4.8, −5.4, and
−5.5. HRMS (electrospray ionization−time-of-flight (ESI−TOF))
m/z calcd for C34H47N6O6Si2 (M + H)+ = 691.3096, found m/z =
691.3097.
Preparation of 3. Compound 6 (64 mg, 0.092 mmol) was
dissolved in THF (1 mL). While stirring, Et3N·3HF (161 mg, 1
mmol) was slowly added. The reaction was stirred at 25 °C overnight.
The reaction was concentrated under vacuum and purified by flash
chromatography on a silica column. Elution with 5% MeOH in DCM
gave 3 as a yellow foam (35.7 mg, 84%). 1H NMR (400 MHz,
MeOH-d4) δ 8.64 (d, J = 6.8 Hz, 2H), 8.48 (d, J = 7.8 Hz, 2H), 8.20
(s, 1H), 7.90 (s, 2H), 6.37 (s, 1H), 4.57 (s, 1H), 4.03 (s, 1H), 3.83
(d, J = 10.1 Hz, 1H), 3.74 (d, J = 10.1 Hz, 1H), 2.80 (s, 1H), and 2.41
(s, 1H). 13C {1H} NMR (101 MHz, DMSO) δ 160.1, 159.8, 140.1,
135.9, 132.1, 132.0, 127.9, 127.4, 122.5, 112.0, 88.1, 83.3, 71.1, and
62.0. HRMS (ESI−TOF) m/z calcd for C22H19N6O6 (M + H)+ =
463.1366, found m/z = 463.1369.
Photolysis of the Precursor and Subsequent HPLC Analysis.
Photolyses were carried out in Pyrex tubes using a Rayonet
photochemical reactor (Southern New England Ultraviolet) equipped
with a merry-go-round apparatus and 16 lamps having a maximum
output at 350 nm. Reaction mixtures (50 μL each) containing 3 (100
μM), thymidine (100 μM) in buffer (10 mM phosphate, pH 7.2), and
BME (10 mM), when appropriate, were photolyzed at room
temperature under aerobic or anaerobic conditions. Samples for
anaerobic reactions were degassed by three freeze−pump−thaw
cycles at 2 m Torr and flame-sealed under vacuum. The reaction
mixtures (including unphotolyzed controls) were analyzed by
reversed-phase HPLC while being monitored at 260 nm. HPLC
was performed on an Phenomenex Luna C-18 column (250 × 4.6
mm) using water and acetonitrile as eluents (1 mL/min) from t = 0 to
1 min holding 3% ACN, from t = 1 to 10 min, from 3 to 28% ACN
linearly, and then from t = 15 to 20 min, from 28 to 97% ACN. The
column was equilibrated for 20 min following a rapid return to the
starting conditions. Peaks corresponding to 2′-deoxyguanosine,
thymidine, 3, and NP eluted at 7.4, 8.3, 15.5, and 16.0 min,
respectively. The peaks were integrated and quantified against the
internal standard thymidine. After collecting the void volume of the
above injections carried out on the C18-reverse phase column, dSP
and dGh analyses were carried out on the UPLC-MS system equipped
with a Thermo Scientific Hypercarb column (130 Å, 5 μm, 2.1 × 100
mm). The gradient (A, 0.1% formic acid; B, acetonitrile, 0.3 mL/min)
was 0% B at t = 0 min, from 0 to 90% B linearly from t = 0 to 10 min
Figure 5. HPLC chromatograms of the photolyses of 3 (0.1 mM)
under anaerobic conditions in a phosphate buffer (10 mM, pH = 7.2)
in the presence of 8-oxodGuo (0.2 mM) detected at 260 nm.
the excited triplet state. Precursor (3) will be a useful tool for
studying the important reactive intermediate, dG(N1-H)•.
EXPERIMENTAL PROCEDURES
■
General Methods. Triethylamine, DMF, and dichloroethane
(DCM) were distilled from CaH2 under Ar or under an appropriate
vacuum. THF was distilled from Na under Ar. All other reagents were
purchased from commercial sources and were used without further
purification unless noted otherwise. All reactions were carried out
under a positive pressure of argon atmosphere and monitored by TLC
on Silica Gel G-25 UV254 (0.25 mm) unless stated otherwise. Spots
were detected under UV light and/or by ethanolic solution p-
anisaldehyde, aqueous solution of ammonium molybdate, ceric
ammonium sulfate, or KMnO4. Column flash chromatography was
performed with Silicycle grade 70−230 mesh, 60−200 μm, 60 Å silica.
The ratio between the silica gel and crude product ranged from 100:1
to 20:1 (w/w). HPLC analyses were carried out using a Waters
system equipped with 515 Pumps, 2489 multiwavelength detector,
and 2707 auto sampler. NMR data were collected using a Bruker
Advance spectrometer. High-resolution mass spectrometry (HRMS)
data were obtained on a Waters Q-TOF mass spectrometer using
electrospray ionization.
Preparation of 6. Compound 528 (135 mg, 0.2 mmol) and
DABCO (22 mg, 0.4 mmol) were dissolved in DMF (2 mL) and
stirred for 30 min. DBU (45 mg, 0.3 mmol) and N-1,8-
hydroxynaphthalimide (4, 213 mg, 1 mmol) were added, and the
reaction was kept at room temperature overnight. The reaction was
quenched by water and extracted by DCM (3 × 10 mL). The
combined organic phase was dried over Na2SO4, filtered, and the
mixture was concentrated under vacuum. The product was purified by
Figure 6. UPLC-MS/MS analysis of the anaerobic photolysis of 3 in the presence of 8-oxodGuo (0.2 mM). (a) Extracted-ion chromatogram (EIC)
of dGh (calculated (M + H) m/z = 274.1146, observed m/z = 274.1143) and (b) EIC of dSp and Sp (calculated (M + H) m/z = 184.0465,
observed m/z = 184.0468).
F
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and holding at 90% B from t = 10 to 12 min. The column was
equilibrated for 3 min following a rapid return to the starting
conditions.
(2) Thapa, B.; Schlegel, H. B. Calculations of pKa’s and Redox
Potentials of Nucleobases with Explicit Waters and Polarizable
Continuum Solvation. J. Phys. Chem. A 2015, 119, 5134−5144.
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Determination of the Quantum Yield for Photoconversion
of 3 in Acetonitrile under Continuous Photolysis. The light flux
of the photoreactor was determined by N-1,8-hydroxynaphthalimide
triflate actinometry in acetonitrile. N-Hydroxy-1,8-naphthalimide
triflate has a 0.23 quantum yield for N−O bond cleavage.26,27
Solutions containing the actinometer (50 μL, 50 mM) were degassed
using 3 freeze−pump−thaw cycles and sealed under high vacuum
prior to photolysis. The extent of reaction of the actinometer was
measured by UV absorbance at 360 nm. The conversion of the
actinometer was 12.4% in 30 s. The percentage transmittance of the
actinometer solution was less than 0.1% after the photolysis.
Therefore, the change of transmittance was negligible during
photolysis. Using eq 1, where nactinometer conversion refers to the amount
of converted actinometer, the light flux was calculated to be (2.5
0.1) × 10−7 Einstein·min−1. Solutions containing 3 (50 μL, 100 μM)
had an OD350 at 1.72. The samples were degassed and photolyzed for
1 min using the calibrated photoreactor. The extent of reaction of 3
was determined by HPLC using thymidine as internal standard. The
conversion of 3 was 30.7 0.9%. The quantum yield for conversion
of precursor 3 was calculated using eq 2, where n3 conversion refers to
the amount of converted precursor 3 and determined to be 0.03.
(4) Kanvah, S.; Joseph, J.; Schuster, G. B.; Barnett, R. N.; Cleveland,
C. L.; Landman, U. Oxidation of DNA: Damage to nucleobases. Acc.
Chem. Res. 2010, 43, 280−287.
(5) Rokhlenko, Y.; Geacintov, N. E.; Shafirovich, V. Lifetimes and
Reaction Pathways of Guanine Radical Cations and Neutral Guanine
Radicals in an Oligonucleotide in Aqueous Solutions. J. Am. Chem.
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(6) Rokhlenko, Y.; Cadet, J.; Geacintov, N. E.; Shafirovich, V.
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nactinometer conversion
light flux =
Φactinometer tphotolysis
(1)
(2)
n3 conversion
Φ3
=
% T
100
1 −
(
(light flux) × t
)
photolysis
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c01095.
■
sı
Imwindelried, P.; Muller, S. N.; Schwitter, U. Cleavage of Single-
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AUTHOR INFORMATION
Corresponding Author
Marc M. Greenberg − Department of Chemistry, Johns Hopkins
University, Baltimore, Maryland 21218, United States;
orcid.org/0000-0002-5786-6118; Phone: 410-516-8095;
Email: mgreenberg@jhu.edu; Fax: 410-516-8420
■
(17) Manetto, A.; Georganakis, D.; Leondiadis, L.; Gimisis, T.;
Mayer, P.; Carell, T.; Chatgilialoglu, C. Independent Generation of
C5’-Nucleosidyl Radicals in Thymidine and 2’-Deoxyguanosine. J.
Org. Chem. 2007, 72, 3659−3666.
(18) Vrantza, D.; Kaloudis, P.; Leondiadis, L.; Gimisis, T.;
Vougioukalakis, G. C.; Orfanopoulos, M.; Gasparutto, D.; Cadet, J.;
Encinas, S.; Paris, C.; Miranda, M. A. Modification of Guanine with
Photolabile N-Hydroxypyridine-2(1H)-thione: Monomer Synthesis,
Oligonucleotide Elaboration, and Photochemical Studies. Helv. Chim.
Acta 2006, 89, 2371−2386.
(19) Kaloudis, P.; Paris, C.; Vrantza, D.; Encinas, S.; Perez-Ruiz, R.;
Miranda, M. A.; Gimisis, T. Photolabile N-hydroxypyrid-2(1H)-one
derivatives of guanine nucleosides: a new method for independent
guanine radical generation. Org. Biomol. Chem. 2009, 7, 4965−4972.
(20) Cadet, J.; Wagner, J. R. Oxidatively generated base damage to
cellular DNA by hydroxyl radical and one-electron oxidants:
Similarities and differences. Arch. Biochem. Biophys. 2014, 557, 47−54.
(21) Cadet, J.; Wagner, J. R. DNA base damage by reactive oxygen
species, oxidizing agents, and UV radiation. Cold Spring Harbor
Perspect. Biol. 2013, 5, No. a012559.
Author
Liwei Zheng − Department of Chemistry, Johns Hopkins
University, Baltimore, Maryland 21218, United States;
orcid.org/0000-0002-1611-2213
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c01095
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the National Institute of General Medical
Sciences (GM-054996, GM-131736) for supporting this
research.
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