5-Carboxamido-5-formamido-2-iminohydantoin, in Addition to 8-oxo-7,8-Dihydroguanine, Is the Major Product of the Iron-Fenton or X-ray Radiation-Induced Oxidation of Guanine under Aerobic Reducing Conditions in Nucleoside and DNA Contexts

Article abstract of DOI:10.1021/acs.joc.5b00689

Exogenously and endogenously produced reactive oxygen species attack the base and sugar moieties of DNA showing a preference for reaction at 2′-deoxyguanosine (dG) sites. In the present work, dG was oxidized by HO^? via the Fe(II)-Fenton reaction or by X-ray radiolysis of water. The oxidized lesions observed include the 2′-deoxynucleosides of 8-oxo-7,8-dihydroguanine (dOG), spiroiminodihydantoin (dSp), 5-guanidinohydantoin (dGh), oxazolone (dZ), 5-carboxamido-5-formamido-2-iminohydantoin (d2Ih), 5′,8-cyclo-2′-deoxyguanosine (cyclo-dG), and the free base guanine (Gua). Reactions conducted with ascorbate or N-acetylcysteine as a reductant under aerobic conditions identified d2Ih as the major lesion formed. Studies were conducted to identify the role of O₂ and the reductant in product formation. From these studies, mechanisms are proposed to support d2Ih as a major oxidation product detected under aerobic conditions in the presence of the reductant. These nucleoside observations were then validated in oxidations of oligodeoxynucleotide and λ-DNA contexts that demonstrated high yields of d2Ih in tandem with dOG, dSp, and dGh. These results identify dG oxidation to d2Ih to occur in high yields leading to a hypothesis that d2Ih could be found from in cells stressed with HO^?. Further, the distorted ring structure of d2Ih likely causes this lesion to be highly mutagenic. (Chemical Equation Presented)

Full text of DOI:10.1021/acs.joc.5b00689

Article
pubs.acs.org/joc
5‑Carboxamido-5-formamido-2-iminohydantoin, in Addition to
8‑oxo-7,8-Dihydroguanine, Is the Major Product of the Iron-Fenton
or X‑ray Radiation-Induced Oxidation of Guanine under Aerobic
Reducing Conditions in Nucleoside and DNA Contexts
Omar R. Alshykhly, Aaron M. Fleming, and Cynthia J. Burrows*
Department of Chemistry, University of Utah, 315 S 1400 East, Salt Lake City, Utah 84112-0850, United States
S
* Supporting Information
ABSTRACT: Exogenously and endogenously produced re-
active oxygen species attack the base and sugar moieties of
DNA showing a preference for reaction at 2′-deoxyguanosine
(dG) sites. In the present work, dG was oxidized by HO• via
the Fe(II)-Fenton reaction or by X-ray radiolysis of water. The
oxidized lesions observed include the 2′-deoxynucleosides of
8-oxo-7,8-dihydroguanine (dOG), spiroiminodihydantoin
(dSp), 5-guanidinohydantoin (dGh), oxazolone (dZ), 5-
carboxamido-5-formamido-2-iminohydantoin (d2Ih), 5′,8-cyclo-2′-deoxyguanosine (cyclo-dG), and the free base guanine
(Gua). Reactions conducted with ascorbate or N-acetylcysteine as a reductant under aerobic conditions identified d2Ih as the
major lesion formed. Studies were conducted to identify the role of O2 and the reductant in product formation. From these
studies, mechanisms are proposed to support d2Ih as a major oxidation product detected under aerobic conditions in the
presence of the reductant. These nucleoside observations were then validated in oxidations of oligodeoxynucleotide and λ-DNA
contexts that demonstrated high yields of d2Ih in tandem with dOG, dSp, and dGh. These results identify dG oxidation to d2Ih
to occur in high yields leading to a hypothesis that d2Ih could be found from in cells stressed with HO•. Further, the distorted
ring structure of d2Ih likely causes this lesion to be highly mutagenic.
H2O + X‐ray → e− + H2O+
INTRODUCTION
aq
(3)
(4)
■
Cellular redox status is a dynamic state that balances reducing
and oxidizing species, and if the balance is thrown off in the cell,
dysfunction arises.1 The hydroxyl radical (HO•) is a powerful
oxidizing species found in the cell and causes redox imbalance
due to its particularly high redox potential (2.31 V vs NHE, pH
7; HO•(+H+)/H2O).2 The Fenton reaction can generate HO•
when a redox-active metal effects cleavage of H2O2, a metabolic
product that can easily diffuse within and between cells.3
Hydrogen peroxide is weakly reactive, but Fe(II) species have
the capability to generate HO• plus Fe(III) via the Fenton
reaction (Reaction 1). Furthermore, reducing agents in the cell
such as ascorbate (Asc) allow this reaction to be catalytic in
iron by reducing Fe(III) back to Fe(II) (Reaction 2).4,5 The
unfortunate events that occurred at Fukushima, Japan in 2011
remind us of the dangers of radiation. The high-energy photons
released upon radioactive decay are capable of yielding HO• via
radiolysis of water (Reactions 3 and 4). The iron-Fenton
reaction and X-ray radiolysis of water represent two methods
for generating hydroxyl radicals that can oxidize biomolecules6,7
such as DNA.4,5,8 DNA damage plays a significant role in aging,
and the development of diseases such as cancer.8
H2O+ → H+ + HO•
DNA is vulnerable to oxidative insults from HO•, and the
damage is particularly deleterious because it can cause
mutations that are heritable to daughter cells. Notable progress
has been made in the discovery of DNA oxidation pathways
resulting from radical oxygen species, such as HO•, during the
past three decades.4,9−12 In DNA, the most susceptible site is
2′-deoxyguanosine (dG) because the guanine heterocycle has
the lowest redox potential (1.29 V vs NHE, pH 7; dG•(−H+)/
dG) among the four DNA bases.13 The nucleoside dG is a
prime target for direct oxidation or electron transfer-mediated
oxidation that can result from HO•.10,13,14 Oxidative damage to
dG yields products that result from the initial product-forming
reaction occurring at one of three sites that include the C5 or
C8 positions of the heterocyclic ring, or the 2-deoxyribose unit
(Figure 1). These products are identified by their characteristic
mass changes from the parent nucleoside dG (M).
Sugar oxidation is a commonly observed reaction pathway of
dG damage resulting from HO•.12 Products observed along this
pathway include 5′,8-cyclo-2′-deoxyguanosine (cyclo-dG)
H2O2 + Fe(II) → Fe(III) + HO• + HO−
(1)
Received: March 27, 2015
2Fe(III) + Asc → 2Fe(II) + dehydroascorbate
(2)
© XXXX American Chemical Society
A
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The Journal of Organic Chemistry
Article
Figure 1. Structures of dG and its oxidation products. Box A: products observed from initial HO• attack at C8. Box B: products observed from initial
HO• attack at C5. Box C: guanine-derived products observed when oxidation occurs on 2-deoxyribose.
diastereomers derived from H• atom abstraction at C5′, and
release of the free base guanine (Gua) after hydroxylation of
C1′. Direct base release in DNA leads to strand breaks due to
the lability of the abasic site so formed.15−17 The yields of these
products are highly dependent on the reaction conditions.12
Base oxidation of dG leads to two lesions resulting from
reaction at C8 that include 8-oxo-7,8-dihydro-2′-deoxyguano-
sine (dOG), observed in high yield under aerobic oxidizing
conditions, or the ring-opened product 2,6-diamino-4-hydroxy-
5-formamidopyrimidine-2′-deoxyribonucleoside (Fapy-dG) ob-
served under anaerobic reducing conditions.18−20 Both dOG
and Fapy-dG are mutagenic compounds found in vivo,9 while
dOG is a biomarker monitored to assay the extent of cellular
oxidative stress.21 Because dOG has a lower redox potential
(0.74 V vs NHE, pH 7; dOG•(−H+)/dOG) than dG,13 it is
readily oxidized to an electrophilic intermediate that reacts with
water at C5 to ultimately yield the hydantoin lesions
spiroiminodihydantoin-2′-deoxyribonucleoside (dSp) and 5-
guanidinohydantoin-2′-deoxyribonucleoside (dGh).8,22−25
These lesions have been observed in vivo and are also highly
mutagentic.26−29
oxidation have identified 5-carboxamido-5-formamido-2-imino-
hydantion-2′-deoxyribonucleoside (d2Ih) as a product from
Fenton chemistry with copper or lead, activation of KHSO5
with nickel or manganese complexes, CO3•‑ oxidations, and the
epoxidizing agent dimethyldioxirane.8,10,35−40 These observa-
tions have led us to the question of whether d2Ih is in fact a
major product from dG oxidation by the hydroxyl radical
generated either from the Fe(II)-mediated Fenton reaction or
by X-ray radiolysis of water.
Toward this goal, we investigated the formation and yield of
dG-oxidation products from the Fe(II)-mediated Fenton
reaction or X-ray radiolysis of water while monitoring the
effect of reductant concentration (ascorbate or N-acetylcys-
teine) on their yields in the nucleoside, short oligodeoxynucleo-
tide, and λ-DNA contexts. These studies identify d2Ih to be a
major guanine oxidation product observed when reactions were
conducted with low millimolar amounts of reductant,
conditions that mimic the presence of reducing agents such
as glutathione, urate, or ascorbate in cells.41 The results of these
studies led to proposed mechanisms for the formation of d2Ih
and to discussions of its possible biological significance.
Additionally, oxidation of dG leads to products resulting
from initial chemistry occurring at the C5 position of the
purine. 2,5-Diaminoimidazolone-2′-deoxyribonucleoside (dIz)
and its hydrolysis product 2,2,4-triamino-2H-oxazol-5-one-2′-
deoxyribonucleoside (dZ) are four-electron oxidation products
resulting from exposure to HO• or one-electron oxidants under
aerobic conditions.8,18,30−34 Recent studies concerning dG
RESULTS AND DISCUSSION
■
Identification of Oxidation Products. The nucleoside
dG (3 mM) was initially chosen for oxidation by the Fe(II)-
mediated Fenton reaction or X-ray irradiation in the absence or
presence of ascorbate (Asc) or N-acetylcysteine (NAC). All
B
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The Journal of Organic Chemistry
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a
Table 1. Absolute Yields for Oxidation of dG by the Fe(II)-Mediated Fenton Reaction
absolute yield (%)
b
b
product
dOG
Fe(II)/H2O2/+Asc/+O2
Fe(II)/H2O2/−Asc/+O2
Fe(II)/H2O2/+Asc/low O2
Fe(II)/H2O2/−Asc/low O2
4.9
5.6
0.4
<0.1
2.7
13.4
0.9
7.1
0.6
0.4
6.6
1.9
dSp
0.9
6.0
3.9
dGh
Gh
<0.1
<0.1
1.5
2.0
1.9
N.D.
N.D.
3.3
N.D.
N.D.
1.0
dZ
d2Ih
cyclo-dG
Gua
OG
0.4
0.2
1.9
1.2
1.5
N.D.
N.D.
N.D.
N.D.
<0.1
dG conversion %
35.8
5.0
20.0
9.9
a
b
Reported values represent the average of three trials that have errors of ∼8% of the value. N.D. = not detected. Low O2 represents a partial
reduction in the O2 concentration by bubbling argon into the sample for 10 min that was later found to be insufficient to completely remove O2.
reactions were conducted with 75 mM NaPi buffer (pH 7.4) at
22 °C. The reaction mixtures were initially analyzed by
reversed-phase UPLC-ESI+-MS. This allowed identification of
guanine (Gua), 8-oxo-7,8-dihydroguanine (OG), dOG, and the
diastereomers of cyclo-dG after comparison to known
standards. The void volume from this analysis was collected
and reanalyzed with a Hypercarb column equipped with an
ESI+-MS detector. This method allowed detection of Gh, dZ,
and the diastereomers of d2Ih, dGh, and dSp. Because dIz
hydrolyzes to dZ during preparation of the void volume, only
dZ was quantified. The Hypercarb column allowed the
separation of the diastereomer products of d2Ih and dSp for
which the absolute configurations for the peaks had been
previously determined.8,42,62 The two diastereomers of dGh are
interconvertible and were not quantified individually.44 Further
support for the identification of d2Ih, dSp, and dZ was
obtained by ESI+-MS/MS fragmentation of the free bases from
in-source fragmentation of the N-glycosidic bond of the parent
nucleosides. The product dGh failed to be fragmented. HRMS
was conducted to further support the identification of each
product (Supporting Information Figures S3−S13).
Product Quantification from the Fe(II)-Mediated
Fenton Reaction. Product quantification was conducted
with reversed-phase HPLC while monitoring peak elution at
240 nm; product peak areas were integrated and normalized
based on the product’s extinction coefficient at 240 nm
(ε240 nm).8 The extinction coefficient for d2Ih has not been
experimentally determined, and due to its instability toward N-
glycosidic bond hydrolysis it was not determined in these
studies.43 However, our previous time-dependent density
functional theory studies on the UV−vis and ECD properties
for dSp and d2Ih allow us to use these previous results to
determine the relative difference in extinction coefficient
between these two compounds at the wavelength moni-
tored.42,43 These calculations determined the ε240 nm for d2Ih
to be ∼30% less than that of dSp; therefore, the ε240 nm value
used to quantify d2Ih was calculated to be 2290 L·mol−1·
cm−1.45 The Fenton reaction was initially conducted with the
Fe(II)/EDTA catalyst and 10 mM H2O2 under aerobic
conditions to give 5% conversion to product. We elected to
conduct reactions to a low overall yield so that primary
oxidation products could be identified and compared. In this
reaction, the highest absolute product yields observed were dZ
(1.5%) and Gua (1.5%) in equal amounts, with the mass
balance completed by low yields of cyclo-dG (0.2%), dOG
(0.4%), dSp (0.9%), and d2Ih (0.4%, Table 1). In the next
reaction, the same conditions were applied with the addition of
2 mM Asc that led to a 7-fold increase in product formation, as
well as a significant change in product distribution. In the
presence of Asc, the major dG product detected was d2Ih
(13.4%) with lower yields of the 2-deoxyribose oxidation
products cyclo-dG (0.9%) and Gua (7.1%), as well as OG
(0.6%) and Gh (<0.1%); also, lower yields of dZ (2.7%), dOG
(4.9%), dSp (5.6%), and dGh (0.4%) were observed (Table 1).
These Asc-dependent studies revealed two key observations:
(1) the reaction yields dramatically increased (7-fold) with Asc
because the Fe(II)/EDTA complex can redox cycle in the
presence of a reductant (Reaction 2), and (2) d2Ih was the
major product observed when Asc was present. Because dSp
and dGh are further oxidation products of the precursor
dOG,22 the yield of d2Ih (13.4%) was compared to the
combined yields of dOG, dSp, and dGh (10.9%), and it was
found that the yield of d2Ih was higher than that of dOG and
its oxidation products.
In the next set of reactions, the role of O2 was studied. First,
a reaction was conducted with dG in the presence of Fe(II)/
EDTA and H2O2 under low O2 conditions achieved by
bubbling argon into the sample for 10 min that was insufficient
to completely remove O2. In this reaction, the overall
conversion of dG to product was 10%, and the major product
observed was dSp (3.9%), with low yields of dOG (1.9%), dGh
(1.9%), d2Ih (1.0%), and the sugar oxidation product cyclo-dG
(1.2%, Table 1). The addition of 2 mM Asc to the low O2
reaction increased the dG conversion by 2-fold (20%) and
changed the product distribution, giving dOG (6.6%) and dSp
(6.0%) in high yield and low yields of d2Ih (3.3%), dGh
(2.0%), and cyclo-dG (1.9%, Table 1). These low O2 studies
indicated the yield of d2Ih was at a minimum under these
conditions. Additionally, the major products observed under
low O2 conditions were dOG and its oxidation product dSp.
Where is Fapy-dG? Many literature sources point to the
presence of Fapy-dG as a guanine oxidation product formed
under anaerobic, reducing conditions.46−48 In our present low
O2 studies, this elusive compound was not observed, even after
exhaustive searching of the LC-MS data. This led us to probe
deeper into the formation of Fapy-dG, using the following
conditions to make this compound from dG by the iron-Fenton
reaction. First, the reaction had to be rigorously purged with
argon for >30 min prior to addition of H2O2 with 2 mM Asc,
and the HPLC mobile phase was changed from running
C
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Figure 2. Effect of reductant concentration on relative product distributions observed from the Fe(II)-mediated Fenton reaction. The reactions were
conducted with 3 mM dG, 10 μM Fe(II)/EDTA, 10 mM H2O2 in 75 mM NaPi buffer (pH 7.4) at 22 °C while varying the concentration of Asc (A)
or NAC (B) from 0−5 mM. Data points represent the average of three trials with an error in each value of 3−10%.
Figure 3. Effect of H2O2 concentration on the absolute product distributions from the Fe(II)-Fenton reaction. The reactions were conducted with 3
mM dG, 10 μM Fe(II)/EDTA, and with either 0.8 mM Asc (panel A) or 3 mM NAC (panel B) in 75 mM NaPi (pH 7.4) at 22 °C, while varying the
concentration of H2O2 from 0−10 mM. Data points represent the average of three trials with an error in each value of 5−11%.
buffered [20 mM NH4OAc (pH 7)] water and MeCN to
unbuffered ddH2O and MeCN. These changes allowed
observation of a broad hump after the void volume in the
reversed-phase HPLC chromatogram with a UV−vis signature
consistent with Fapy-dG (λmax = 270 and 218 nm),46 and a
defined peak that eluted after dG with a similar UV−vis
profile.46 The broad hump was consistent with literature
reports because Fapy-dG exists as anomers of two different
sugar configurations.46,47 LC-ESI+-MS of all these new peaks
only found masses consistent with the Fapy free base, as well as
a peak with mass Fapy + 18 (Figure S24). These observations
identify that Fapy-dG is not stable to laboratory manipulation
as are d2Ih, dOG, dSp, or dGh, and it appears to be sensitive to
nucleophilic buffers in the HPLC mobile phase, e.g., NH4OAc.
Further test reactions that had argon bubbled through them for
a few seconds up to 1 min before H2O2 addition did not furnish
the Fapy peaks. The Fapy peaks only appeared after purging
the system with argon for >30 min. To reiterate, in cases with
low O2 and Asc present, dOG was the major product observed,
and when O2 was at ambient concentrations with Asc present,
the major product was d2Ih. These observations identify Fapy-
dG to be very challenging to obtain, as it is only observed under
extreme anoxic conditions (∼100% argon and ∼0% O2),
conditions under which cells cannot survive. If Fapy-dG was
formed in a cell, detection of the nucleoside would be
problematic due to the hydrolytic instability of the N-glycosidic
bond. This later feature is in contrast to d2Ih, dOG, dSp, and
dGh that are all at least stable enough to allow their
characterization under very mild conditions (22 °C in water
buffered at pH 7).
Finally, Fapy-dG has a low redox potential (1.1 V vs NHE)47
rendering it subject to further oxidation; however, this is also
the case for dOG (0.7 V vs NHE)13 that has an even lower
redox potential, and we detect and quantify this intermediate
species and its further oxidation products dSp and dGh. Thus,
the conditions outlined do not simply over-oxidize Fapy-dG
causing its loss. We conclude that Fapy-dG is not a major
contributor to the product distribution of guanosine oxidation
by a hydroxyl radical under cellularly relevant conditions.
Reducing-Agent-Dependent Studies for the Iron-
Mediated Fenton Reaction. In cells, two main small-
molecule reductants are found in high concentrations, ascorbate
and glutathione, found at concentrations of 0.8−3 mM and 1−
10 mM, respectively.41 Knowing that d2Ih yields increased
when Asc was present during the Fe(II)-mediated Fenton
oxidation of dG (Table 1), we then studied the role of
increasing reductant concentrations on the relative yields of the
products. In addition, the nature of the reductant was studied,
in which we chose N-acetylcysteine (NAC) as a model for
glutathione. Note that protection of the amine group of
cysteine with an acetyl group prevents amine adduct formation
with dG oxidation intermediates.49,50
Titration of Asc from 0−5 mM into the Fe(II)-mediated
Fenton oxidation of dG provided the following trends in the
relative product distributions (Figure 2A): (1) As the Asc
concentration increased, the reaction yield increased, support-
ing the role of Asc in allowing the iron catalyst to redox cycle
D
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between Fe(II) and Fe(III) and thereby increasing the
production of HO• (Reactions 1 and 2). (2) The amount of
2-deoxyribose oxidation decreased (∼10%) as the concen-
tration of Asc increased. This observation negates the
involvement of Asc in sugar oxidation, and it is likely that
Asc inhibits this pathway. (3) The relative yields of dOG, dSp,
and dGh slightly increased (∼6%) with more Asc. (4) The Asc
concentration was shown to be critical in the relative yields of
the C5 products, dZ and d2Ih. When Asc was not present, the
major C5-oxidation product was dZ (also a major product
overall); however, as Asc was titrated into the reaction, the
relative yield of dZ decreased and d2Ih increased until it was
the major product of the reaction at Asc >2 mM (Figure 2A).
Within the range of physiological Asc concentrations (1−3
mM),41 d2Ih was always the major oxidation product detected
(Figure 2A). Moreover, when the study was conducted with
variable NAC concentrations, the same trends were observed
(Figure 2B). Again, the most notable observation is that d2Ih
was the major oxidation product observed in reactions
conducted with >2 mM thiol concentrations (Figure 2B),
even when considering a comparison of d2Ih (∼37%) with the
combined yields of dOG, dSp, and dGh (∼30%, Figure 2A and
2B with 2 mM reductant).
H2O2 Concentration-Dependent Studies for the Iron-
Mediated Fenton Reaction. In the next set of studies, dG
oxidation product distribution versus the H2O2 concentration
(0−10 mM) was monitored with a static amount of reductant
(0.8 mM Asc or 3 mM NAC, Figure 3A and 3B, respectively).
Key observations from these studies include the following: (1)
As expected, the reaction yield increased as more H2O2 was
added to the reaction (Supporting Information, Figures S16
and S17). (2) With increased H2O2 concentrations, the flux of
HO• increased, and the relative yield of dSp increased at the
expense of dOG. This observation supports dOG as the
intermediate leading to dSp.10,11 (3) Both dZ and d2Ih
increased as a function of H2O2 concentration (0−10 mM);
moreover, the amount of d2Ih increased 4-fold when Asc was
the reductant while dZ increased 3-fold (Figure 3A). In the
H2O2-dependent studies with NAC, d2Ih increased 13-fold
while dZ increased 3-fold when H2O2 increased from 0 to 10
mM (Figure 3B). These studies demonstrate that the yield of
d2Ih was maximal under conditions that boost formation of
HO•.
Product Quantification from X-ray-Mediated Oxida-
tion of dG. Next, oxidation of dG was conducted with an X-
ray source at a dose rate of 25 Gy/min for 30 min (total dose =
750 Gy). Initial studies without a reductant gave a 45%
conversion of dG to product. The major oxidation products
observed were dZ (14.7%), dSp (10.8%), and Gua (12.4%) and
in low absolute yields were d2Ih (2.9%), dOG (1.4%), and
dGh (1.4%). Next, Asc (2 mM) was added to the reaction to
give the following changes in the yields. (1) The absolute
conversion of dG to product decreased with Asc (2.5-fold), as
expected, because Asc quenches radical reactions.51 (2) The
major products were now d2Ih (6.3%), dSp (3.9%), and Gua
(4.1%), with the mass balance completed by low yields of dZ
(1.1%), dOG (2.5%), cyclo-dG (0.5%), and dGh (<0.1%). The
most striking observation was that d2Ih was the major
oxidation product when Asc was present during X-ray mediated
oxidations; however, this is in contrast to studies without Asc
where dZ was the major oxidation product quantified (Table
2). When considering the yield of d2Ih (6.3%) in comparison
to the combined yields of dOG, dSp, and dGh (6.4%), the
Table 2. Absolute Yields for Oxidation of dG by X-ray
Radiolysis of Water
a
Absolute Yield (%)
product
X-ray +Asc/+O2
X-ray −Asc/+O2
dOG
dSp
2.5
3.9
0.2
<0.1
1.1
6.3
0.5
4.1
0.2
1.4
10.8
1.4
dGh
Gh
0.5
dZ
14.7
2.9
d2Ih
cyclo-dG
Gua
OG
0.1
12.4
N.D.
dG conversion %
18.8
45.3
a
Reported values represent the average of three trials, and the error in
each value was 4−10%. N.D. = not detected.
initial split between d2Ih and the C8 products was nearly equal
with X-ray irradiation when reductant was present. Because the
X-ray reactions required the reaction vessel to be open to the
atmosphere, studies were not conducted under anaerobic
conditions.
Reducing-Agent-Dependent Studies for the X-ray
Mediated Oxidations. Reactions with and without Asc
suggest a major role for the reductant in the product
distributions for X-ray mediated oxidations (Table 2). There-
fore, studies were conducted with the reductant Asc or NAC
titrated (0−5 mM) into the X-ray-mediated reaction mixture
while monitoring the product yields (Figure 4A and 4B). From
these studies, the following observations were made in X-ray-
mediated oxidations: (1) As the reducing agent concentration
increased, the reaction yield significantly decreased (Supporting
Information, Figures S19 and S21). (2) The relative yield of
dOG increased (6-fold) as a function of reductant concen-
tration (Figure 4A and 4B), while the yield of dSp decreased
(1.5-fold) and dGh decreased (3-fold). This result supports the
conclusion that the reductant quenched the further oxidation of
dOG to the hydantoins. (3) The yields of the sugar oxidation
products were not significantly changed by the increase in
reductant concentration (Figure 4A and 4B). (4) The reductant
had the most dramatic effect on the C5-pathway products, dZ
and d2Ih. When the reductant was not present, dZ was the
major product and d2Ih was observed in very low yield;
however, when Asc or NAC was increased to relevant
concentrations, d2Ih was the major oxidation product observed
with very little dZ detected.
Total X-ray Dose Effect on dG Oxidation Product
Distributions. In the next set of reactions, dG was irradiated
with X-rays at a rate of 25 Gy/min while varying the time (i.e.,
total dose) and keeping the Asc concentration static (2 mM).
As the total dose of X-rays increased from 25 to 750 Gy, the
following trends were observed. (1) As expected, the overall
reaction yield increased (Supporting Information Figure S22).
(2) The yield of dSp and dGh increased at the expense of dOG
with increasing X-ray dose. (3) The yield of sugar chemistry
increased with increasing X-ray dose. (4) The yield of dZ and
d2Ih both increased as a function of the X-ray dose, and d2Ih
was always the major C5-oxidation pathway product, as well as
being the major product of the reaction overall (Figure 5).
Proposed Pathways Leading to dG Oxidation Prod-
ucts. Under all of the conditions studied, products derived
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Figure 4. Effect of reductant concentration on the dG-oxidation product yield from the X-ray reaction. The reactions were conducted with 3 mM dG
in 75 mM NaPi (pH 7.4) at 22 °C with a 30 min exposure to 25 Gy/min X-ray source (total dose = 750 Gy) while varying the concentration of Asc
(panel A) or NAC (panel B) from 0−5 mM. Data points represent the average of three trials with an error in each value of 3−10%.
from sugar oxidation were observed. Oxidation at the 5′-carbon
yields the 5′ carbon-centered radical that attacks C8 of the
heterocyclic ring of dG to furnish an intermediate radical that
loses another electron and proton to furnish cyclo-dG (Scheme
1). Oxidation at the 1′, 2′, 3′, 4′, or 5′ carbons also yields a
carbon-centered radical that reacts with O2, and many of these
pathways ultimately cause cleavage of the N-glycosidic bond
releasing Gua (Scheme 1).12,15 Evidence of these sugar
oxidation pathways was determined by quantification of Gua
release. The yield of cyclo-dG in the Fenton reaction was
maximal under anaerobic reducing conditions and was observed
to be at a minimum under aerobic nonreducing conditions
(Table 1). In contrast, base release to give Gua was observed to
be maximal under aerobic and reducing conditions and not
Figure 5. Effect of X-ray irradiation time on dG-oxidation product
distributions. The reactions were conducted with 3 mM dG, 2 mM
Asc in 75 mM NaPi (pH 7.4) at 22 °C while varying the exposure time
observable under anaerobic conditions (Table 1). These results
further support the role of O2 in effecting base release from 2-
deoxyribose oxidation.12 Moreover, this reaction was not
sensitive to the presence of the reductant. However, under
anaerobic conditions the 5′-carbon radical was not trapped by
(0−30 min) to an X-ray source that was delivered at a rate of 25 Gy/
min. Data points represent the average of three trials with an error in
each value of 3−10%.
a
Scheme 1. Proposed Pathway for Products Derived from Sugar Oxidation
a
For the sake of brevity, only oxidation of the 1′-carbon leading to Gua is shown.
F
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O2, but rather added into the heterocyclic ring at C8 leading to
cyclo-dG, and again this reaction was not sensitive to the
presence of the reductant. Also observed in very low yield were
OG and Gh, two products that are derived from further
oxidation of Gua (Table 1). The observation of Gh and not Sp
under these conditions (pH 7.4 and 22 °C) is consistent with a
previous observation that oxidations of the free base OG only
give Gh and not Sp.52 In the X-ray-mediated oxidation of dG,
O2-dependent studies could not be conducted; therefore, only
product dependence on the reductant was studied. In this
study, the products derived from sugar oxidation were not
greatly affected by the presence of the reductant (Table 2).
Other products resulting from oxidation of the 2-deoxyribose
sugar in dG were not observed by LC-ESI+-MS (Figure S3).
Oxidation of dG at the C8 position yields dOG and Fapy-
dG.4,10 Mechanistically, these products form when HO• adds at
the C8 carbon to yield 8-HO-dG• (Scheme 2). This
Furthermore, as the oxidant flux increases, the yield of dOG
decreases due to further oxidation to dSp.
The low redox potential of dOG renders this nucleoside
labile toward further oxidation to yield the hydantoins dSp and
dGh (Scheme 3).22,54 Two-electron oxidation of dOG followed
Scheme 3. Proposed Pathway for Products Derived from
Further Oxidation of dOG
Scheme 2. HO•-Mediated Oxidation of dG at C8 Leading to
dOG and Fapy-dG
by water attack at C5 yields 5-HO-dOG that bifurcates along
two pathways to either dSp or dGh.22,43,44,54 Acyl migration to
dSp dominates under conditions of higher pH (pH > 5.8) and
unencumbered contexts such as nucleosides, single-stranded
DNA, and G-quadruplexes, while the yield of dGh is highest at
low pH (pH < 5.8) or sterically encumbered contexts such as
double-stranded DNA.22,43,44,54,55 These and previous obser-
vations explain why dSp was the major hydantoin observed in
all unencumbered nucleoside studies. As previously stated, the
yield of dOG was highest when the reductant was present at
relevant concentrations (Figures 2 and 4). In contrast, the yield
of dSp was highest in the absence of the reductant. This
observation supports dOG being the stable intermediate that
leads to dSp, as further oxidation of dOG to dSp was quenched
with added reductant (Figures 2 and 4). These data, in which
dOG was obtained in greater yield than the hydantoins under
conditions with physiologically relevant reductant, further
explain why concentrations of dOG are generally much greater
than hydantoins in vivo.26,56
intermediate can be further oxidized to yield dOG, or be
reduced and ring opened to yield Fapy-dG (Scheme 2).4,10
Under all conditions, dOG was observed; however, we did not
detect Fapy-dG in these studies without exhaustive removal of
O2 (Figure S24). In both the Fenton and X-ray oxidations, the
yield of dOG was at a maximum when the reductant was
present, which appears to be inconsistent with previous
results;20 however, the current studies were conducted under
aerobic conditions (Tables 1 and 2), while the previous studies
were conducted under anaerobic conditions. Because dOG can
undergo further oxidation to hydantoin products (see below),22
the presence of the reductant suppresses the further oxidation
and addresses why dOG concentrations are highest under
oxidations conducted with the reductant. In conclusion, HO•-
mediated oxidation of dG via Fe(II)-Fenton chemistry and X-
ray irradiation effects oxidation at C8 of dG to yield dOG as the
dominant product along this pathway under low oxidant flux
with O2 and the reductant present (Figures 3A, 3B, and 5).
Oxidations of dOG have detected a four-electron oxidation
product dehydroguanidinohydantoin-2′-deoxyribonucleoside
(dGhox), or formally a six-electron oxidation product of dG
(Scheme 3).57 This compound has a short half-life and
G
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Scheme 4. Proposed Pathway for Oxidation of dG Followed by Initial Reaction at C5 Leading to dZ
Scheme 5. Proposed Pathways for d2Ih Product Derived from C5 Pathway Oxidation
24
Next, dG• couples with O2 to ultimately yield 5-HOO-dG
•−
decomposes to yield oxaluric acid that further hydrolyzes to
liberate oxalate and ultimately 2′-deoxyribosyl-urea.58 These
analytical conditions allow identification of oxaluric acid
(unpublished result from our laboratory); however, under the
current HO•-mediated oxidations, neither dGhox nor oxaluric
acid were detected. In these studies, low product conversion
was maintained, preventing hyperoxidation of dG to dGhox.
The products dZ and d2Ih result from oxidation of dG
followed by initial product forming chemistry occurring at C5.
Previous HO•-mediated oxidations of dG detected dZ as a
major product under aerobic and nonreducing conditions,
which we observed in these studies (Tables 1 and 2).59,60
Mechanistically, dZ was proposed to form from one-electron
oxidation of dG (dG•+/dG•(−H+), pKa ∼3.9)55 to a neutral
that is the intermediate susceptible to reduction. When the
reductant was not present, 5-HOO-dG decomposes to yield
dIz; however, when the reductant was present, the hydro-
peroxyl group can be reduced to the alcohol 5-HO-dG. Next,
5-HO-dG undergoes acyl migration to reduced-dSp (dSpred)
that hydrates at C8 and ring opens to yield d2Ih (Scheme 5A).
This proposed mechanism provides a role for O2 and the
reducing agent in the formation of d2Ih that was observed in
the product distribution studies (Figures 2 and 4). The
proposed mechanistic steps from 5-HO-dG to d2Ih were
previously proposed from dG oxidations with KHSO5 catalyzed
by a Mn−porphyrin complex.36,61
Because d2Ih was also observed under anaerobic conditions
(Table 1) there must be an alternative pathway for its
formation that is not O2 dependent. In Scheme 5B, such a
mechanism is proposed in which HO• adds to C5 yielding 5-
HO-dG• that proceeds through a second one-electron
oxidation and deprotonation to yield 5-HO-dG; acyl migration
and hydration then yield d2Ih (Scheme 5B). This second
pathway provides a route to d2Ih under anaerobic and
nonreducing reaction conditions. The alternative pathway
must be a minor reaction channel to d2Ih, because added
reductant significantly increases d2Ih formation supporting the
mechanism in Scheme 5A.
•−
radical that couples with O2/O2 to ultimately yield a
hydroperoxy intermediate (5-HOO-dG). Next, 5-HOO-dG
decomposes through a multistep process to dIz followed by
hydration to dZ (Scheme 4).54,56,57 Additional support for the
role of O2/O2•− in the product defining step leading to dZ was
observed in the reactions under anaerobic conditions, for which
dZ was not detected (Table 1). Further, as the reducing agent
was titrated into the reactions, the yield of dZ dramatically
decreased, an observation consistent with a previous report.59
When the oxidations were conducted under aerobic
conditions with relevant concentrations of the reductant (Asc
or NAC), d2Ih was the major product detected (Figures 3, 4,
and 5). In Scheme 5 a mechanism is proposed to explain this
observation. Formation of HO• via the Fe(II)-Fenton reaction
or X-ray radiolysis of water (Reactions 1 − 4) effects the one-
electron oxidation of dG followed by proton loss to yield dG•.
Product formation upon oxidation of dG initially occurs on
the sugar leading to base release or cyclo-dG (Scheme 1), on
C5 of the heterocyclic base leading to d2Ih and dZ (Schemes 4
and 5), or on C8 of the heterocyclic base leading to Fapy-dG
and dOG (Scheme 2), in which dOG is further oxidized to dSp
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and dGh (Scheme 3). Inspection of the product distributions
allows ranking the initial dG site of reactivity toward oxidation
under each condition. The focus will be on reactions conducted
under aerobic conditions with and without the addition of Asc
(Tables 1 and 2). The iron-Fenton reaction without the
addition of Asc focused on the sugar, C5, and C8 carbons in a
ratio of 1.3:1.5:1.0, respectively, with the C5 products showing
the highest yield. A comparison of the C5 and C8 product
distributions show that C5 products are ∼150% of the C8
products. When Asc (2 mM) was added to the mixture, the
ratio of reactivity was 1.0:1.9:1.3 for the sugar, C5, and C8
carbons, respectively (Table 1). The addition of Asc to the
iron-Fenton reaction suppressed oxidation of the sugar, and
initial reaction at the C5 was still greater than C8. More
interestingly, C5 products were still ∼150% more than the C8
products. This observation is consistent with the proposed
mechanistic role of Asc in product formation, in which it
reduces intermediate species that have already reacted at C5 or
C8 (Scheme 5).
extinction coefficients to obtain their yields (Figure S24).
These limitations in product analysis prevent an ideal
comparison; however, we can use these data to understand
context-dependent product distributions leading to C5
products (2Ih and Z) vs C8 products (OG, Sp, and Gh)
when iron-Fenton or X-ray mediated oxidations were
conducted with Asc (2 mM) present under aerobic reaction
conditions.
The overall context-dependent trends were very similar for
iron-Fenton and X-ray irradiation reactions (Figure 6 A and B).
The X-ray mediated oxidation of dG without Asc yielded
sugar, C5, and C8 products in a 1.0:1.4:1.0 ratio, respectively
(Table 2). Initial reactivity at C5 was the dominant pathway,
and it was ∼140% greater than the C8 pathway, similar to that
observed for the iron-Fenton mediated oxidation of dG. When
Asc was added to the mixture the ratio of reactivity at the sugar,
C5, and C8 sites on dG was 1.0:1.5:1.4, respectively. Overall,
Asc decreased the sugar oxidation products (Table 2) and the
C5 and C8 products were found in nearly the same yield. The
reason C5 and C8 products were observed in similar yields has
to do with dZ. The presence of Asc dramatically diminished the
yield of dZ in the X-ray mediated oxidations, leading to less C5
products and similar yields of C5 and C8 products. These
comparisons support a greater level of oxidation occurring at
the heterocyclic ring of the dG nucleoside compared to the
sugar particularly with Asc present.
Use of a Hypercarb column allowed separation of the d2Ih
and dSp diastereomers for which the absolute configurations
and elution order on this HPLC column are known.42,62 In the
current studies, the R and S isomers of d2Ih were observed in a
1:2 ratio, respectively, for both oxidation reactions studied,
while the R and S isomers of dSp were observed in a 1:1 ratio.
Furthermore, these ratios did not change under any of the
reaction conditions studied. The observation that the d2Ih
isomers were not in a 1:1 ratio points to steric hindrance during
the defining point of the reaction that determines product
stereochemistry and was likely caused by the 2-deoxyribose
sugar. In contrast to this result, the diastereomer ratio of d2Ih
found from the copper-mediated Fenton oxidation was 2:1.8 In
the other d2Ih studies, the diastereomer identity and ratios
were not stated.35,36,38−40
In the last study, the products resulting from base oxidation
of dG in oligonucleotides of known sequence in single-
(ODN1) and double-stranded (ODN2) contexts were
determined, as well as products observed from λ-DNA. The
product analysis was achieved using HF to hydrolyze the bases
from the sugar−phosphate backbone. This approach prevented
the quantification of sugar oxidation leading to base release.
Because of this limitation, only base oxidation products are
compared. Further, products resulting from oxidation of other
nucleotides, leading to thymine glycol, for example, were
observed but were not quantified; masses consistent with
guanine cross-links with thymine and cytosine were observed,
but their yields are not reported due to a lack of established
Figure 6. Context-dependent yields of dG base oxidation products
from the iron-Fenton and X-ray irradiated samples. The ODN and λ-
DNA distributions were obtained after HF hydrolysis of the oxidized
samples. In the ODN and λ-DNA samples, products were also
observed from oxidation of the other nucleotides. Because of the HF
hydrolysis to liberate the oxidized free bases, the extent of sugar
oxidation leading to free base could not be determined.
The relative yield of Z diminished dramatically when
comparing oxidations between nucleoside to ODN and DNA
contexts. In the duplex contexts no Z was observed, an
observation similar to previous studies of the added reductant
during the oxidations.52 The OG free base was observed in all
contexts and comprised the smallest relative yield in the ODN
and DNA contexts and largest yield in the nucleoside studies
from both iron-Fenton and X-ray oxidations. The yield of Sp
and Gh showed strong context-dependent yields, in which Sp
was greatest in nucleoside and single-stranded contexts and Gh
was observed to be greatest in duplex contexts. This
observation is consistent with previous reports.8,43 Lastly, the
relative yield of 2Ih remained nearly the same in all three
contexts (∼40−50%, Figure 6A and B). Without complete
mass balances for oxidations in the ODN and DNA contexts, it
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a
Table 3. Comparison of the Current Results to Other Reports
initial reaction at C8
initial reaction at C5
products
initial reaction at 2-deoxyribose
sugar
oxidants
HO• (radiolysis)
dOG
dSp/dGh
dIz/dZ
d2Ih
reference
59,60
+
+
++
+
N.D.
++
++
++
++
++
++
++
++
++
+
•−
CO3
+
+
+
39
40
35
37
8
Pb(II)/H2O2
+
+
+
+
NiCR/KHSO5
N.D.
N.D.
+
+
N.D.
N.D.
+
N.D.
N.D.
+
DMDO
N.D.
+
Cu(I)/H2O2
Dicopper(II)-complex
Mn-TMPyP/KHSO5
Fe(II)-EDTA/H2O2/Asc or NAC
X-ray/Asc or NAC
N.D.
N.D.
+
N.D.
+
N.D.
+
N.D.
+
64
61
+
+
+
current study
current study
+
+
+
+
a
(++) a major product, (+) a minor product, (N.D.) not detected or not reported.
cannot be definitively stated that d2Ih is the major oxidation
product of dG; nonetheless, d2Ih is clearly a major product
observed from oxidation of dG in the nucleoside, single- and
double-stranded ODN, and λ-DNA contexts. This observation
supports the possibility of d2Ih formation in the cellular
context.
two-electron oxidation products of dG that have not been
previously detected in oxidations with HO• generated by the
Fe(II)-mediated Fenton reaction or X-ray radiolysis of H2O.
The high yields of d2Ih observed and the observation that this
lesion is highly prone to piperidine cleavage in a DNA
oligomer, unlike dOG,65 lead to a hypothesis that oxidations in
DNA imposed by electron transfer agents yield C5 oxidation
products (i.e., d2Ih or dZ) and not the C8 oxidation product
dOG.66 Oxidations in the context of single- and double-
stranded ODNs and λ-DNA yielded d2Ih as a product of dG
oxidation in significant yield that was competitive with the yield
of dOG. More importantly, the current results highlight d2Ih as
a major product observed in reactions that include relevant
amounts of reductant in the presence of O2, suggesting that
d2Ih should be investigated more closely for its biochemical
properties.
The current results are compared to literature studies that
reported d2Ih as shown in Table 3. The Ball laboratory
exclusively observed d2Ih when dG was exposed to the
epoxidizing reagent dimethyldioxirane (DMDO), and they
confirmed the structure of d2Ih via complementary NMR
methods.37,63 The Meunier laboratory observed d2Ih (M+34)
when dG was oxidized with Mn-TMPyP/KHSO5.36,61 Studies
in the Karlin and Rokita laboratories observed a product mass
in high yield consistent with d2Ih (M+34) when an
oligodeoxynucleotide was allowed to react with a dicopper-
(II)-complex.38,64 In our laboratory, d2Ih was detected in high
yield with the NiCR/KHSO5 system, and in studies utilizing
the copper-mediated Fenton oxidation of dG.8,35 Further, the
Bohme laboratory conducted the lead-mediated Fenton
oxidation of dG to yield d2Ih as the major product.40 In all
of the metal-catalyzed studies, the transition metal was
proposed to play a role in the product-forming step leading
to d2Ih. However, studies conducted in the Shafirovich
EXPERIMENTAL PROCEDURES
■
Fenton Reaction. A 200-μL solution of dG (3.0 mM, 0.6 μmoles,
0.16 mg) in a buffer (75.0 mM NaPi, pH 7.4) was mixed with Fe(II)/
EDTA (0.10 mM, 0.02 μmoles, 0.008 mg), H2O2 (10.0 mM, 2 μmoles,
0.5 mg), and Asc or NAC (2.0 mM, 0.40 μmoles, 0.03 mg) and
incubated for 1 h at 22 °C. The Fe(II)/EDTA complex was freshly
made by mixing Fe(NH4)2(SO4)2 and Na2EDTA in a 1:2 ratio 30 min
prior to reaction. The Fe(II)/EDTA complex was removed by column
chromatography using ion-exchange resin prior to HPLC analysis
following the method outlined below.
•−
laboratory observed d2Ih during CO3 oxidations of dG,
demonstrating that a transition metal was not required for d2Ih
formation.39 In the current report, d2Ih was the major product
of “free” HO•-mediated oxidation of dG with relevant amounts
of reductant under aerobic conditions. The X-ray studies
demonstrate once again that d2Ih can be formed under
conditions that do not require the involvement of a transition-
metal catalyst.
X-ray Irradiation. A 200-μL solution containing dG (3.0 mM, 0.6
μmoles, 0.16 mg) in buffer (75.0 mM NaPi, pH 7.4) and Asc or NAC
(2.0 mM, 0.40 μmoles, 0.03 mg) was irradiated with an X-ray RS 2000
biological research irradiator source at 22 °C for variable times (1−30
min). The dose rate was 25 Gy/min.59 This solution was analyzed by
the method outlined below.
Oligodeoxynucleotide and DNA Oxidations. Single-stranded
ODN1 or double-stranded ODN2 were oxidized in 20 mM NaPi (pH
7.4) with 100 mM NaCl at 37 °C. Iron-Fenton oxidations were
conducted in a 200-μL reaction volume with ODN (100 μM, 0.01
μmoles, 0.05 mg) to which were added Fe(II)EDTA (0.10 mM, 0.02
μmoles, 0.008 mg), Asc (2.0 mM, 0.40 μmoles, 0.03 mg), and H2O2
(1.0 mM, 0.2 μmoles, 0.05 mg); the reaction was allowed to proceed
for 30 min. The stock solutions of Fe(II)EDTA, Asc, and H2O2 were
all freshly prepared. To irradiate the ODNs, they were placed in the
same buffer system as the iron-Fenton reaction followed by X-ray
irradiation with a dose rate of 25 Gy/min for 30 min (total dose = 750
Gy). For the λ-DNA oxidations, everything was identical to the ODN
oxidations with the exception that the λ-DNA (1 μM, 0.1 nmol, 0.03
mg) was different.
CONCLUSIONS
■
The current studies monitored oxidation products from dG
resulting from HO• generated via the Fe(II)-mediated Fenton
reaction or X-ray radiolysis of water. Products resulting from
oxidation and initial reaction at the sugar, C5, or C8 carbons
were quantified (Figures 2−5). Under aerobic conditions
without a reductant, the major products observed include dZ,
dSp, and sugar oxidation leading to Gua. Under anaerobic
reaction conditions the major product observed was dSp. When
reactions were conducted with cellularly relevant (mM)
amounts of the reductant (Asc or NAC) and O2, the major
product observed in all cases was d2Ih. The isomers of d2Ih are
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Hydrolysis of Oxidized Oligodeoxynucleotides and λ-DNA.
The oxidized ODN or λ-DNA samples were hydrolyzed to the free
bases for HPLC analysis. The hydrolysis was performed on the
oxidized and lyophilized DNA by adding 50 μL of 70% HF in pyridine
for 30 min at 37 °C. After the reaction, the excess HF was neutralized
by adding 1 mL of ddH2O and 80 mg of CaCO3 to the sample. The
insoluble salts were removed by centrifugation, and the supernatant
was then lyophilized to dryness. Next, the lyophilized samples were
dissolved in ddH2O and submitted to LC-MS and HPLC analysis as
described for the nucleoside studies below.
ACKNOWLEDGMENTS
■
The authors are grateful to Dr. James Muller (University of
Utah) for his assistance with mass spectrometry and to the
National Institutes of Health for financial support (R01
CA090689).
REFERENCES
■
(1) Sies, H. Klin. Wochenschr. 1991, 69, 965−968.
(2) Breen, A. P.; Murphy, J. A. Free Radic. Biol. Med. 1995, 18, 1033−
1077.
Product Identification. Product identification was initially
achieved by UPLC-ESI+-MS (100 mm × 2.1 mm, 1.7 μm) and
UPLC-ESI+-MS with a Hypercarb column (100 mm × 2.1 mm, 5
μm). Then, each compound was HPLC purified for further structural
analysis. The following masses were observed: Gua m/z [M + H]+
calcd 152.1, found 152.1. OG m/z [M + H]+ calcd 168.1, found 168.1.
dOG m/z [M + H]+ calcd 284.2, found 284.1. R and S diastereomers
of cyclo-dG m/z [M + H]+ calcd 266.2, found 266.1. Gh m/z [M +
H]+ calcd 158.1, found 158.1. (S)-d2Ih and (R)-d2Ih42 m/z [M + H]+
calcd 302.3, found 302.1; ESI+-MS/MS m/z [M + H]+ lit.37 186, 158,
and 141; found 186, 158, and 141. HRMS (ESI-TOF) m/z [M + Na]+
calcd for C5H7N5O3Na 208.0447, found 208.0449. The HRMS value
was obtained on the free base 2Ih, due to the nucleoside’s instability
toward acid. dGh m/z [M + H]+ calcd 274.3, found 274.1; HRMS
(ESI-TOF) m/z [M + Na]+ calcd for C9H15N5O5Na 296.0971, found
296.0980. (S)-dSp and (R)-dSp62 m/z [M + H]+ calcd 300.2, found
300.1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C10H13N5O6Na
322.0764, found 322.0761; ESI+-MS/MS m/z [M + H]+ lit.22 184,
156, 141, 113, 99, and 86; found 184, 156, 141, 113, 99, and 86. dZ m/
z [M + H]+ calcd 247.2, found 247.1; HRMS (ESI-TOF) m/z [M +
Na]+ calcd for C8H14N4O5Na 269.0862, found 269.0870; ESI+-MS/
MS m/z [M + H]+ lit.67 247, 203, and 131; found 247, 203, and 131
(Figures S3−S13, Supporting Information). The dGh diastereomers
were characterized by NMR,44 dZ was characterized by NMR,30 cyclo-
dG was characterized by X-ray crystallography,68 the dSp diaster-
eomers have been characterized by X-ray crystallography69 and
NMR,70 and the diastereomers of d2Ih have been previously
characterized by NMR.37
Product Quantification. The oxidation products from the Fe(II)-
Fenton reaction or X-ray irradiation reaction were quantified by C18
reversed-phase HPLC and Hypercarb HPLC. First, the reaction
mixture was injected on a reversed-phase HPLC (250 mm × 4.6 mm,
5 μm) to quantify Gua, OG, dOG, and cyclo-dG. The void volume
from this run was collected, lyophilized to dryness, and then dissolved
with the Hypercarb column starting mobile phase (0.1% acetic acid).
Next, the reconstituted void volume was injected on a Hypercarb
column (150 mm × 4.6 mm, 5 μm) to quantify Gh, (R)-d2Ih, (S)-
d2Ih,42 dGh, (R)-dSp, (S)-dSp,62 and dZ. Product peaks were
quantified by their absorbance intensity at 240 nm followed by
normalization of these intensities by each compound’s extinction
coefficient at 240 nm. The values for ε240 nm (ddH2O) are dG 14 080,
dOG 14 300, cyclo-dG 14 080, Gua 14 080, OG 14 300,15 dSp 3280,22
d2Ih 2290,8,35 dGh and Gh 2410,54 and dZ 1780.67 All values are in
units of L mol−1 cm−1.
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(4) Burrows, C. J.; Muller, J. G. Chem. Rev. 1998, 98, 1109−1152.
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ASSOCIATED CONTENT
* Supporting Information
UPLC-ESI+-MS, HPLC-ESI+-MS, ESI+-MS/MS, reversed-
phase HPLC, Hypercarb HPLC, UV−vis spectra, and product
distribution tables. The Supporting Information is available free
of charge on the ACS Publications website at DOI: 10.1021/
acs.joc.5b00689.
■
S
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AUTHOR INFORMATION
Corresponding Author
*E-mail: burrows@chem.utah.edu.
Notes
■
The authors declare no competing financial interest.
K
DOI: 10.1021/acs.joc.5b00689
J. Org. Chem. XXXX, XXX, XXX−XXX

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J. Org. Chem. XXXX, XXX, XXX−XXX