Independent Generation and Time-Resolved Detection of 2′-Deoxyguanosin-N2-yl Radicals

Article abstract of DOI:10.1002/anie.202005300

Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HO^.) has long been believed to react with 2′-deoxyguanosine (dG) generating 2′-deoxyguanosin-N1-yl radical (dG(N1-H)^.) via addition to the nucleob

Full text of DOI:10.1002/anie.202005300

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Accepted Article
Title: Independent Generation and Time-Resolved Detection of 2'-
Deoxyguanosin-N2-yl Radical
Authors: Liwei Zheng, Xiaojuan Dai, Hongmei Su, and Marc M.
Greenberg
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202005300
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10.1002/anie.202005300
Angewandte Chemie International Edition
RESEARCH ARTICLE
Independent Generation and Time-Resolved Detection of 2'-
Deoxyguanosin-N2-yl Radical
Liwei Zheng,†[a] Xiaojuan Dai,†[b] Hongmei Su,*[b] and Marc M. Greenberg*[a]
[a]
Dr. L. Zheng, Prof. M. M. Greenberg
Chemistry
Johns Hopkins University
3400 N. Charles St., Baltimore MD 21218 (United States of America)
E-mail: mgreenberg@jhu.edu
X. Dai, Prof. H. Su
Chemistry
Beijing Normal University
[b]
[†]
Beijing 100875 (P. R. China)
These authors contributed equally to this work.
E-mail: hongmei@bnu.edu.cn
Supporting information for this article is given via a link at the end of the document.
Abstract: Guanine radicals are important reactive intermediates in
DNA damage. Hydroxyl radical (HO•) has long been believed to react
with 2'-deoxyguanosine (dG) generating 2'-deoxyguanosin-N1-yl
radical (dG(N1-H)•) via addition to the nucleobase -system and
subsequent dehydration. This basic tenet was challenged by an
alternative mechanism, in which the major reaction of HO• with dG
was proposed to involve hydrogen atom abstraction from the N2-
amine. The 2'-deoxyguanosin-N2-yl radical (dG(N2-H)•) formed was
proposed to rapidly tautomerize to dG(N1-H)•. We report the first
independent generation of dG(N2-H)• in high yield via photolysis of 1.
dG(N2-H)• is directly observed upon nanosecond laser flash
photolysis (LFP) of 1. The absorption spectrum of dG(N2-H)• is
corroborated by DFT studies, and anti- and syn-dG(N2-H)• are
resolved for the first time. The LFP experiments showed no evidence
for tautomerization of dG(N2-H)• to dG(N1-H)• within hundreds of
microseconds. This observation suggests that the generation of
dG(N1-H)• via dG(N2-H)• following hydrogen atom abstraction from
dG is unlikely to be a major pathway when HO• reacts with dG.
was recently proposed to arise by tautomerization of dG(N2-H)•
(Scheme 1).[7] We have resolved this problem by using near-UV
photolysis of a synthetic precursor to dG(N2-H)• in conjunction
with time-resolved spectroscopy, time-dependent DFT
calculations and product studies.
Introduction
Scheme 1. Generation of guanine radicals by hydroxyl radical (HO•).
Nucleic acid oxidation is central to human health. For instance,
it is a factor in aging and in the development of cancer.[1] DNA is
also the molecular target for a variety of cancer treatments. Many
of these methods involve one electron oxidation of DNA, with
ionizing radiation being the most common.[2] Ionizing radiation
damages DNA directly by ionizing DNA and indirectly by ionizing
water, which generates hydroxyl radical (HO•), a highly reactive
DNA damaging species.[3] 2'-Deoxyguanosine (dG) is the most
readily oxidized of the 4 native nucleosides and is also a primary
contributor to electron transfer in one-electron oxidized DNA.[4]
Consequently, the reactive intermediates produced upon dG
oxidation, and their reactivity, have been the focus of important
theoretical studies and experimental investigations for the past 30
years.[5] Pulse radiolysis has been used extensively to
characterize the early, rapid dG oxidation events that are
complete on the sub-millisecond timescale.[5c] dG(N1-H)• is the
major and thermodynamically most stable intermediate generated
by reaction of dG with HO• (Scheme 1).[6] However, the
mechanism(s) by which dG(N1-H)• is formed is controversial and
It is widely accepted that HO• reacts with dG yielding dG(N1-
H)• via an addition-elimination mechanism. HO• adds to C4, C5
and C8 atoms of guanine. C4-OH (Scheme 1) is proposed to be
the major product, accounting for 60-70% of the reactions.[6a]
Computational studies indicate that upon barrierless addition of
HO•, C4-OH produces dG(N1-H)• via loss of hydroxide to form an
ion pair, followed by N1-deprotonation.[5b] Calculations predict that
ion pair formation encounters ~6.5 kcal/mol barrier and is the rate
determining step. N1-deprotonation within the ion pair is
kinetically and thermodynamically favored (>7 kcal/mol) over the
N2-position. The calculated energy difference between the
radicals is basis set dependent, but dG(N1-H)• is 2-4 kcal/mol
more stable than dG(N2-H)•.[8]
The HO• pathway to dG(N1-H)• through C4-OH was challenged
by a series of pulse radiolysis experiments carried out on various
guanine derivatives.[7] The authors posited that HO• preferentially
1
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RESEARCH ARTICLE
abstracts the N2-hydrogen atom, instead of adding to the -bond
to generate C4-OH (Scheme 1). Initially formed dG(N2-H)• was
proposed to rearrange to the more stable dG(N1-H)• with a < 30
s half-life at 298 K and an activation energy of ~5.5 kcal/mol. The
feasibility of the hydrogen atom abstraction of this alternative
mechanism was corroborated by DFT calculations.[5b] Pulse
radiolysis, in conjunction with spectroscopic detection (and other
measurements), is a powerful approach for studying reactive
intermediate chemistry. However, one limitation is that multiple
reactive intermediates can be produced. This can potentially
complicate analysis, particularly if reactive intermediates have
overlapping spectral properties. To simplify the examination of
purine radicals and resolve mechanistic conflicts in the
aforementioned studies, we designed a photochemical precursor
(1) that generates a single purine intermediate, dG(N2-H)•. We
thoroughly examined the reactivity of this radical by product
analysis following UV-photolysis of 1 and time-resolved LFP
experiments that corroborate each other. In addition to refuting
the sub-millisecond tautomerization of dG(N2-H)•, we also
distinguished the anti- and syn-conformers of dG(N2-H)• for the
first time.
We previously generated dG(N2-H)• and 2'-deoxyadenosin-
N6-yl radical (2) from the corresponding diphenyl hydrazines (e.g.
3, Scheme 2).[9] However, photochemical conversion of 3 to
dG(N2-H)• is too inefficient for laser flash photolysis examination
of dG(N2-H)•. More recently, we reported on a method for
generating 2'-deoxyadenosin-N6-yl radical (2) from a ketone
precursor (4, Scheme 2).[10] Upon photolysis, 4 undergoes Norrish
Type I photocleavage followed by rapid -fragmentation. Using
acetone as triplet photosensitizer greatly accelerated the
conversion of 4 and allowed us to obtain the spectrum of 2.
However, photosensitization of 3 by ketones was not attempted,
because photodissociation of tetraphenylhydrazine occurs from
the excited singlet.[11] Furthermore, we anticipated that the
ketones, which photo oxidize 8-oxodGuo, would do the same to
the more readily oxidizable 3.[9, 12] We rationalized that 1 would
yield dG(N2-H)• via the analogous cascade of reactions that 4
undergoes upon photolysis, and could also be sensitized by
ketones (Scheme 2). The synthetic approach to.1 was strongly
Results and Discussion
Design and synthesis of a photochemical precursor (1) for
dG(N2-H)•.
Scheme 2. Photochemical generation of purine radicals from synthetic precursors.
Scheme 3. Synthesis of dG(N2-H)• precursor 1.[a]
2
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RESEARCH ARTICLE
influenced by that of 4 and started from previously reported 5
is approximately twice as high as when a reducing agent is
added (Figure S6). This is consistent with the proposal that the
radical precursor can serve as a reducing reagent for dG(N2-
H)•, a pathway that will be significant in laser flash photolysis
(LFP) experiments described below.
13]
(Scheme 3).[10a,
Substitution of the bromide in 5 by
hydroxylamine (6) was slower than the analogous reaction in
the synthesis of 4 due to the increased electron density of
guanine. The formation of 6 required higher temperature than
that for the dA analogue, and dioxane was substituted for THF,
because of its higher boiling point. Introduction of the hindered
ketone 8 using previously reported conditions, in which NaH
was used as base led to the formation of an undesired product.
Subsequently, the substitution was successfully carried out
using Cs2CO3 as base. The desired ketone (1) was then
obtained via standard debenzylation and desilylation
conditions.
Table 2. Effect of acetonitrile on the conversion of 1 (0.1 mM) within 30 min
in the presence of acetophenone (1% v/v).
Acetonitrile (% v/v)
% Conversion of 1[a]
28 ± 2
20[b]
50[b]
90[c]
58 ± 1
Photochemical generation of dG(N2-H)• and product
studies.
95 ± 1
With an eye on utilizing 1 as a source of dG(N2-H)• in DNA,
we carried out photolyses in Pyrex vessels using lamps whose
maximum output is at 350 nm. Although the max for 1 occurs
in a far shorter region (max = 260 nm,  = 1.22 x 104 M-1s-1 in
H2O) than where these lamps emit, the absorption band tails
above 300 nm (Figure S5). The quantum yield for
disappearance of 1 under these conditions, measured using 2-
hydroxy-2-methylpropiophenone as an actinometer, was
similar ( = 1.8  10-3) to that of 4 ( = 1.5  10-3).[10a, 14]
However, the weaker absorbance of 1 above 300 nm resulted
in less efficient photochemical conversion than 4, such that
only ~10% of the ketone was consumed following 8 h direct
irradiation. Inspired by the sensitization used during the
photolysis of 4, we used acetone (2% v/v,150 mM) to sensitize
the reaction and achieved increased conversion to 62.4 ± 0.9%
after 8 h in the presence of PhSH as reducing agent. The yield
of dG and mass balance were high when Fe2+ or PhSH were
used as a reducing agent (Table 1). In contrast to 2, -
mercaptoethanol (BME) also effectively trapped dG(N2-H)•
producing dG.[10a] We attribute this difference to the fact that
guanine is more electron-rich than adenine. The corresponding
nitrogen radical (dG(N2-H)•) is less electrophilic than 2, and
encounters lower energy barriers when reacting with
electronegative thiol hydrogen atom donors. These
observations are consistent with our hypothesis regarding the
polarity matching between hydrogen atom donors and neutral
purine radicals.[10a] Purine electronic properties also manifest
themselves when photolysis is carried out in the absence of a
reducing agent. Under these conditions the mass balance and
dG yield decrease almost two-fold but the conversion rate of 1
[a] aAverage ± std. dev. of 3 experiments. [b] Acetonitrile in phosphate buffer
(10 mM, pH = 7.2). [c] Acetonitrile in water.
Although acetone photosensitizes 1, comparison to the
sensitizer’s effect on
4
indicated that photochemical
conversion would be too low for LFP studies.[10a] Consequently,
we considered photolysis in the presence of other
photosensitizers. Acetophenone was a promising candidate
due to its efficient intersystem crossing, long triplet lifetime, and
relatively high triplet energy.[15] Anaerobic photolyses were
carried out in the presence of acetophenone (1% v/v, 86 mM),
and the consumption of the precursor was significantly
accelerated. The sensitization efficiency is proportional to the
percentage of acetonitrile in phosphate buffer (Table 2). The
source of this solvent effect is uncertain but it is unlikely that it
is due to the decreased energy of π, π* excited triplet state of
acetophenone in more polar solvent mixtures.[16] In the
presence of 100 mM BME, the photolysis of 1 quantitatively
yielded dG, indicating that sensitization was not detrimental to
the fidelity of the photochemistry.
dG(N2-H)• characterization by laser flash photolysis and
DFT studies.
In light of the observation that photosensitized photolysis of
1 is a high fidelity source of dG(N2-H)•, this system was used
to directly observe the latter via transient absorption
spectroscopy. Rich transient features are observed upon
nanosecond pulses (355 nm) of solutions of 1 (1 mM) and
acetophenone (30 mM) in aqueous buffer (pH 7.0)/acetonitrile
(1:1, v:v). Following 355 nm excitation, the triplet
acetophenone absorption band at ~340 nm is immediately
observed.[17] This transient decays within 4 µs (Figure 1a, S7),
and is accompanied by a build-up of a strong absorption band
with maxima at 610 nm and 650 nm. We attribute these
observations to photosensitization of 1 by triplet acetophenone
and the resulting reactions that give rise to dG(N2-H)•. The
timescale for the growth of dG(N2-H)• is consistent with laser
flash photolysis and computational studies on the formation of
2 via -fragmentation following Norrish Type I photocleavage
of 4 (Scheme 2).[10a] The transient feature centered at 610 nm
is consistent with the previously reported absorption of N1-
MedG(N2-H)• from N1-MedG.[5c, 7d, 18] The red-shifted peak at
650 nm has not been reported in relevant studies.
Table 1. Product studies upon anaerobic photolysis of 1 in the presence of
acetone (2% v/v) as sensitizer.[a]
Reducing Agent (mM)
Fe2+ (10)
% Yield dG[b]
85 ± 2
% Mass Balance[b]
90 ± 1
PhSH (10)
BME (10)
89 ± 3
93 ± 1
87 ± 2
91 ± 2
None[c]
55 ± 3
65 ± 1
None[d]
35 ± 2
44 ± 3
[a] [1] = 0.1 mM. [b] Average ± std. dev. of 3 experiments. [c] Anaerobic
photolysis. [d] Aerobic photolysis
3
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2) indicate that neither absorbs above 600 nm (Table S2). The
observation that the growth and decay kinetics of the 650 nm
peak are essentially identical to that of the 610 nm peak (Figure
1), suggested that they belong to very similar species. Given
that product studies indicate that 1 produces dG(N2-H)• with
high fidelity, we postulated that the peaks at 610 and 650 nm
belong to different conformational isomers of dG(N2-H)• in
which the remaining N2 hydrogen is either syn or anti with
respect to the guanine N3 atom (Figure 2). (The naming
convention is that utilized by Sevilla.[19]) The optical spectra
(Figure 3a) for these two conformers in analogues lacking the
deoxyribose ring (syn-, anti-Gua(N2-H)•) were calculated by
TDDFT-B3LYP/6-311++G(d,p) in vacuum and under PCM. To
compare with experiments, the TDDFT calculated absorption
maxima for guanine radicals usually require being red-shifted
by 40 – 70 nm.[6b] The calculated spectra for each Gua(N2-H)•
conformer features an intense absorption band above 600 nm
and weaker band in the UV (Figure 3a). The calculations
reproduce the main feature of the experimental spectra for
dG(N2-H)•. In addition, it is found that the max of the main
absorption band > 600 nm is different for these two conformers
(Table S2). Under PCM solvation model, the calculated max
(after adding 60 nm) for the syn-conformer (632 nm) is red-
shifted relative to the anti-conformer (616 nm). In vacuum, the
calculated max (after adding 40 nm) for syn-Gua(N2-H)• is 652
nm and that for the
Figure 1. Transient UV-vis absorption spectra of
1 (1 mM) and
acetophenone (30 mM) in aqueous buffer (pH 7.0) /acetonitrile (1:1, v:v)
upon 355 nm laser flash photolysis in anaerobic conditions (A.) within the
first 4 μs (B.) 100 μs. Inset: Normalized early time kinetics traces for the
transients at 340 nm, 610 nm, 650 nm.
A variety of possible molecules responsible for the peak at
650 nm were considered. The assignment of this peak to the
triplet excited state of 1, or the aminoxy alkyl radical
intermediate 10 resulting from Norrish Type I cleavage
analogous to that produced from 4 were ruled out by
calculations. DFT calculations on 10 and the triplet excited
state of 9 (the analogue of 1 lacking a 2'-deoxyribose, Figure
Figure 2. The B3LYP/6-311++G(d,p)//PCM optimized geometries of syn- and anti-Gua(N2-H)•, as well as syn- and anti-9 (the analogues for precursor 1 lacking of
the deoxyribose ring).
4
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is also consistent with calculations on N9-methyl guanine
radicals.[20-21]
Our
calculations
based
on
the
DFT/B3LYP/6-
311++G(d,p)//PCM method show that syn-Gua(N2-H)• is ~ 0.1
eV (~ 2.3 kcal/mol) lower in energy than the anti-conformer,
which is in general agreement with the literature (3.0 kcal/mol
at the level of B3LYP/6-31G(d)//PCM).[19] In the presence of
five explicit waters and under PCM solvation model (Figure S8),
the B3LYP/6-311++G(d,p) calculated energy difference
between the syn- and anti-Gua(N2-H)• is further reduced to
0.064 eV (~ 1.5 kcal/mol). The small energy difference means
that the two conformers of dG(N2-H)• may coexist in aqueous
solution. Previously, N1-MedG(N2-H)• was produced by
7d, 18]
deprotonation of the corresponding radical cation.[5c,
Given the small energy difference of the two conformers and
low energy barrier for deprotonation (~ 4.75 kcal/mol), the anti-
and syn-conformational isomers are expected to form in
approximately equal amounts from this intermediate.[22] The
dominant absorption peaks (> 600 nm) of anti-dG(N2-H)• and
syn-dG(N2-H)• are predicted to overlap significantly with
comparable intensity (Figure 3a). Consequently, it is expected
that the two peaks of the anti- and syn- dG(N2-H)• may not be
resolved in the spectrum containing both conformers in
approximately equal amounts. The spectra of N1-MedG(N2-
H)• produced by one-electron oxidation and deprotonation,
where only a single broad peak centered at ~ 630 nm are
consistent with this.[5c, 7d, 18]
Figure 3. TDDFT-B3LYP /6-311++G(d,p) //PCM calculated absorption
spectra of guanine radicals in water after red-shifting by 60 nm (A.): anti-
(black) and syn- (red) Gua(N2-H)•; (B.) Gua(N1-H)•. Lengths of sticks
correspond to the relative oscillator strengths of electronic transitions. Each
electronic transition is convoluted using a Gaussian function with half width
at half maximum (HWHM) of 0.33 eV.
anti-conformer is 612 nm. The theoretically predicted
absorption wavelengths for the two conformers match the
experimental spectrum obtained from the photolysis of 1
(Figure 1b), indicating that the peak with max = 650 nm is
ascribable to syn-dG(N2-H)•, which is partially resolved from
anti-dG(N2-H)• max = 610 nm. Experiments in G-quadruplex
DNA corroborate the predicted spectral dependence upon
dG(N2-H)• conformation.[20] When dG(N2-H)• is produced from
the deprotonation of dG•+ in G-quadruplex structure, the
observed spectrum centered at ~600 nm is consistent with
anti-dG(N2-H)•. Selective formation of anti-dG(N2-H)• in the G-
quadruplex is attributed to preferential syn-N2 deprotonation,
which does not disrupt Hoogsteen hydrogen bonding.[20] The
predicted conformational dependence of guanine radical max
Figure 4. Experimental (black dots) transient UV-vis absorption spectrum at
4 µs of dG(N2-H)• with the fitted spectrum (red line), composed of two
individual Gaussian bands peaking at ~ 610 (blue line) and ~ 650 nm (dark
yellow line).
Table 3. Double exponential fitting results for the decay kinetics at 610 nm and 650 nm in the absence and presence of thiols.
Reducing agent (mM)
None
610 nm 1 (μs)
37.5 ±1.1
610 nm 2 (μs)
298.9 ± 4.7
161.2 ± 6.6
151.5 ± 4.2
650 nm 1 (μs)
34.0 ±1.0
650 nm 2 (μs)
322.4 ± 4.7
166.5 ± 2.6
165.5 ± 4.4
BME (100)
GSH (10)
34.9 ± 2.7
30.4 ± 1.9
38.3±1.3
35.1± 1.9
5
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maximum signal (4 µs) is reproduced by combining two broad
peaks with max at ~ 610 and 650 nm (Figure 4). Dividing these
two peak areas by their respective molar absorption
coefficients (estimated using the calculated oscillator strengths
in Table S2) suggests that the observed spectrum is the result
of an ~ 2:1 mixture of syn-dG(N2-H)• relative to anti-dG(N2-H)•.
The decays of the 610 and 650 nm bands produced upon
sensitized photolysis of 1 (1 mM) in the absence of additional
reducing agent were fitted to double-exponentials and
exhibited comparable lifetimes (Figure 5, Table 3),
corroborating the proposal that the bands are attributable to
syn- and anti-dG(N2-H)•. The shorter lifetime decay constants
change little with the addition of reducing agent, but are
dependent on the concentration of the radical precursor. We
attribute this decay pathway to the reduction of dG(N2-H)• by
its precursor (1), a process that was detected in product
studies (Table 1). This bimolecular process is more prominent
in the laser flash photolysis experiments, which are carried out
at significantly higher concentrations of 1 and greater photon
fluxes. In contrast, the slower decay process is affected by the
addition of glutathione (GSH) or BME. Attributing the change
in the lifetimes of the slower decay constants for the transient
at 610 nm and 650 nm to reduction of dG(N2-H)• by the thiols
indicates that BME (~2.7
– 2.9 x
104 M-1s-1) reacts
approximately 10-fold more slowly with the nitrogen-centered
radical than does GSH (~3.0 – 3.2 x 105 M-1s-1). These rate
constants are considerably slower than what would be
expected for reaction with a carbon-centered radical.[23]
However, they are consistent with reactions of other nitrogen-
centered radicals that are conjugated to electron accepting
24]
substituents, such as a purine ring (e.g. 2).[10a,
These
Figure 5. Decay kinetics traces and biexponential fitting for the 610 nm (a);
and 650 nm (b) bands obtained from the photosensitized photolysis of 1
(1mM) in the absence and presence of thiols of BME (100 mM) and GSH
(10 mM).
radicals and dG(N2-H)• are electron deficient and kinetically
mismatched for reaction with thiols.
dG(N2-H)• also features a less intense absorption band at ~
370 nm, which is evident upon diminution of the transient
absorption of triplet acetophenone after 4 µs. Subsequently,
the band at 370 nm decays at comparable rates as those at
610 nm and 650 nm (Figure S10).[7] This observation is
inconsistent with the proposed tautomerization of dG(N2-H)• to
dG(N1-H)•. Pulse radiolysis experiments indicate that dG(N1-
H)• absorbs strongly in this region.[5c] TDDFT-B3LYP/6-
31++G(d,p)//PCM calculations indicate that dG(N1-H)• should
absorb more strongly in this region than dG(N2-H)• (Figure 3,
Table S2). These spectral features for the two radicals are
affirmed by other computational studies.[6b] Furthermore,
radiolysis studies showed that dG(N1-H)• decays relatively
slowly with a lifetime of ~0.07 s.[25] Consequently, if dG(N2-H)•
tautomerized to dG(N1-H)• with the reported first-order rate
constant of 2.3 x 104 s-1 (t1/2 < 30 s), the absorption in the 370
nm region would have maintained its intensity or even
increased slightly during the time that the longer wavelength
In contrast to the generation of N1-MedG(N2-H)• via
deprotonation of N1-MedG•+, the conformation dG(N2-H)•
generated via sensitized photolysis of 1 is controlled by the
conformation of the precursor, which also exists in anti- and
syn-conformations (Figure 2). B3LYP/6-311++G(d,p)//PCM
calculations of the analogue lacking a 2'-deoxyribose indicate
that syn-8 is more stable than anti-8 by 0.21 eV (~4.83
kcal/mol). The more abundant syn-1 is expected to result in a
greater amount of syn-dG(N2-H)• than anti-dG(N2-H)•.
Moreover,
DFT
calculations
at
DFT/B3LYP/6-
311++G(d,p)//PCM level showed that the energy barrier for the
transition between syn-dG(N2-H)• and anti-dG(N2-H)• is 0.89
eV (20.5 kcal/mol) (Figure S9). This significant energy barrier
suggests that the interconverison between the two conformers
is kinetically infeasible on the LFP experiment timescale.
These results are consistent with a recent report by Sevilla in
which rotation of the N2-H group in N1-MedG(N2-H)• from 0 to
60° with respect to the purine ring also indicated a high
rotational barrier.[6b] Consequently, the significant rotational
barrier and the preference for generating syn-dG(N2-H)• by 1
results in a partially resolved spectrum of the two conformers.
(Figure 1). The transient spectrum for dG(N2-H)• with
bands for dG(N2-H)• decay (Figure 1, 3).[7d] [6b]
Finally, the
.
observation that the intense absorption features of dG(N2-H)•
at 610 nm and 650 nm exist for hundreds of microseconds,
provide additional evidence against rapid tautomerization
(Figure S11).
Conclusion
6
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On account of their being common intermediates in DNA
damage, the structure and reactivity of purine radicals have
garnered significant interest. UV-photolysis of appropriately
designed precursors is a common approach for generating
homogeneous solutions of these and other DNA radicals.[26]
The generation and reactivity of dG(N2-H)• has been a
contentious issue, in part because it has only been produced
using radiolysis, which may not produce homogeneous
solutions of the radical. To address this issue, we developed a
photochemical precursor (1) that produces dG(N2-H)• in high
yield, as evidenced by product studies. Photosensitization by
acetophenone enabled using 1 as a high fidelity source of
dG(N2-H)• in laser flash photolysis experiments, where the
distinct spectral features of anti- and syn-dG(N2-H)• are
resolved . LFP affirmed product studies, showing that dG(N2-
H)• is reduced by precursor 1. dG(N2-H)• also reacts with thiols,
albeit significantly more slowly than carbon-centered radicals.
Importantly, photochemical generation of dG(N2-H)• from 1
enabled us to address the proposed microsecond timescale
tautomerization of dG(N2-H)• to dG(N1-H)•. Chatgilialoglu,
Steenken and Sevilla had independently reported spectra that
Acknowledgements
We are grateful for support from the National Institute of General
Medical Sciences (GM-054996 and GM-131736). H.S. thanks the
funding support from the National Natural Science Foundation of
China (21933005, 21727803 and 21425313). We thank Amit
Adhikary and Michael Sevilla for helpful discussions.
Keywords: DNA damage • guanine oxidation • radical
reactivity • laser flash photolysis • oxidative stress
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7d]
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10.1002/anie.202005300
Angewandte Chemie International Edition
RESEARCH ARTICLE
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8
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Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
dG(N2-H)• and dG(N1-H)• are major reactive species produced upon guanine oxidation. dG(N2-H)• was independently generated
from 1. dG(N2-H)• reacts significantly more slowly with thiol reducing agents than alkyl radicals. Contrary to previous reports, there is
no evidence that dG(N2-H)• rearranges to dG(N1-H)• on the sub-millisecond timescale
9
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