5,6-dihydropyrimidine peroxyl radical reactivity in DNA

Article abstract of DOI:10.1021/ja412562p

Nucleobase radicals are a major family of reactive species produced in DNA as a result of oxidative stress. Two such radicals, 5-hydroxy-5,6- dihydrothymidin-6-yl radical (1) and 5,6-dihydrouridin-6-yl radical (5), were independently generated within chemically synthesized oligonucleotides from photochemical precursors. Neither nucleobase radical produces direct strand breaks or alkali-labile lesions in single or double stranded DNA. The respective peroxyl radicals, resulting from O₂ trapping, add to 5′-adjacent nucleobases, with a preference for dG. Distal dG's are also oxidatively damaged by the peroxyl radicals. Experiments using a variety of sequences indicate that distal damage occurs via covalent modification of the 5′-adjacent dG, but there is no evidence for electron transfer by the nucleobase peroxyl radicals.

Full text of DOI:10.1021/ja412562p

Article
pubs.acs.org/JACS
Open Access on 02/28/2015
5,6-Dihydropyrimidine Peroxyl Radical Reactivity in DNA
Joanna Maria N. San Pedro and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: Nucleobase radicals are a major family of reactive
species produced in DNA as a result of oxidative stress. Two such
radicals, 5-hydroxy-5,6-dihydrothymidin-6-yl radical (1) and 5,6-
dihydrouridin-6-yl radical (5), were independently generated
within chemically synthesized oligonucleotides from photo-
chemical precursors. Neither nucleobase radical produces direct strand breaks or alkali-labile lesions in single or double
stranded DNA. The respective peroxyl radicals, resulting from O2 trapping, add to 5′-adjacent nucleobases, with a preference for
dG. Distal dG’s are also oxidatively damaged by the peroxyl radicals. Experiments using a variety of sequences indicate that distal
damage occurs via covalent modification of the 5′-adjacent dG, but there is no evidence for electron transfer by the nucleobase
peroxyl radicals.
Scheme 1
INTRODUCTION
■
Hydroxyl radical (•OH) is a major reactive intermediate
produced when water is exposed to ionizing radiation. Its
reactions with DNA constitute the “indirect effect” of ionizing
radiation and account for as much as 85% of the damage
imparted upon the molecular carrier of genetic information in
cells.1,2 Fe·EDTA (and similar metal complexes), an agent that
is widely used to probe the structure and binding interactions of
DNA (and RNA), relies upon its ability to cleave nucleic acids
by producing •OH.3 Strand scission by •OH is attributed to
hydrogen atom abstraction from the C4′- and C5′-nucleotide
positions.4 However, a large number of studies in which
ionizing radiation is used to generate •OH indicate that
hydrogen atom abstraction from the carbohydrate components
of nucleic acids accounts for as little as 7% of the overall
reactions.2,5,6 The major pathway is believed to involve •OH
addition to the π-bonds of the nucleobases. The subsequent
reactivity of the nucleobase radicals and their respective O2
trapping products has been a topic of considerable interest to
understand the ultimate chemical effects of ionizing radiation
on nucleic acids. Pyrimidine •OH radical adduct reactivity has
received greater attention than the corresponding purine
reactive intermediates. Radiation scientists have employed a
variety of sophisticated and clever methods to extract
information from experiments in which •OH is generated in
the bulk medium (solution, thin films, and glasses) resulting in
the formation of multiple reactive intermediates. We and others
are studying reactive intermediates in nucleic acids by
independently generating individual species from photo-
chemical precursors incorporated at defined sites in chemically
synthesized oligonucleotides.7−9
abstraction may occur from the (2′-deoxy)ribose of another
nucleotide (internucleotidyl) or intramolecularly (intranucleo-
tidyl). When internucleotidyl hydrogen atom abstraction occurs
at a nucleotide within several base pairs of the original site at
which •OH reacted the aggregate damage constitutes a
clustered lesion. Clustered (complex) lesions also result from
reaction of a nucleotide (peroxyl) radical with another
nucleotide’s nucleobase and are believed to play an important
role in the cytotoxicity of ionizing radiation due to their
inefficient repair compared to isolated lesions.15−19 Tandem
lesions are a subset of clustered damage and describe
modification on contiguous nucleotides.20,21 Pyrimidine
nucleobase peroxyl radicals have been proposed to produce
tandem lesions involving adjacent 2′-deoxyguanosines (dG) by
directly oxidizing the purine as well as adding to the
purine.21−23 More recently, pyrimidine peroxyl radicals were
proposed to initiate electron transfer (hole migration) within
DNA by oxidizing dG.24 Herein we describe how we have
Strand scission requires that the spin be transferred from the
nucleobase to the carbohydrate backbone of the nucleic acids.
Dihydropyrimidine radicals and/or their respective peroxyl
radicals have been proposed to induce strand scission by
abstracting hydrogen atoms from the carbohydrate components
of RNA and to a lesser extent DNA.6,10−14 Hydrogen atom
Received: December 10, 2013
Published: February 28, 2014
© 2014 American Chemical Society
3928
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
a
examined these issues by independently generating the major
•OH radical adduct of thymidine (1, Scheme 1) and the
structurally related species, 5 (Scheme 2). (Please note that the
same descriptor is used for a compound as a monomer or when
it is within a biopolymer.)
Scheme 3
Scheme 2
a
Key: (a) Ac2O, pyridine; (b) Et3N·3HF; (c)DMTCI, pyridine; (d)
phosphitylation.
Lesions Resulting from Hydrogen Atom Abstraction
from the Sugar Backbone. Hydrogen atom abstraction from
the 2′-deoxyribose ring produces direct strand breaks and/or
NaOH labile oxidized abasic sites, depending upon the position
from which the hydrogen is removed.32,33 Previous studies
established that monomeric 1 generated from 8 does not
abstract hydrogen atoms from its own 2′-deoxyribose ring.26,27
There also is no evidence for intranucleotidyl or internucleo-
tidyl hydrogen atom abstraction by 5,6-dihydro-2′-deoxyuridin-
6-yl radical (5) when it is generated in DNA.15 However, the
respective peroxyl radical (7) formed by O2 trapping of 5
abstracts the C1′-hydrogen atom from the 5′-adjacent
nucleotide.15,18,19 Using 2 as a precursor, we observed that
the reactivity of 5-hydroxy-5,6-dihydrothymidin-6-yl radical (1)
paralleled that of 5 in the absence of O2. Specifically, anaerobic
photolyses of duplex DNA containing 2 (5′-32P-15 or 5′-32P-
16) failed to produce any direct strand scission or alkali-labile
sugar lesions at the nucleotides bonded to the 5′-phosphate of
the radical precursor.31
The reactivities of 5-hydroxy-5,6-dihydrothymidin-6-yl radi-
cal (1) and 5 were not as similar to one another when O2 was
present. Like 5, direct strand scission did not occur when 1 was
produced from irradiation of 5′-32P-15 or 5′-32P-16 under
aerobic conditions. However, as opposed to experiments
involving 5, mild alkaline treatment (0.1 M NaOH, 37 °C, 30
min) of the photolysates in which 5-hydroxy-5,6-dihydrothy-
midin-6-yl radical (1) was formed did not produce any strand
scission at the respective 5′-adjacent nucleotides, suggesting
that 3 did not effect internucleotidyl hydrogen atom
abstraction.31
RESULTS AND DISCUSSION
■
Independent Generation of 5,6-Dihydropyrimidin-6-
yl Radicals within Oligonucleotides. Hydroxyl radical is
electrophilic and preferentially adds to the more electron-rich
C5-position of pyrimidines.2 In addition to being a synthetically
expedient analogue of 1, radical 5 is the formal product of
hydrogen atom addition to 2′-deoxyuridine, which is also
generated from ionization of water. 5,6-Dihydro-2′-deoxyur-
idin-6-yl (5) was previously generated in oligonucleotides via
Norrish Type I photocleavage of 6 (Scheme 2).18,19,25 The C5-
hydroxyl radical adduct of thymidine (1) was independently
generated via photoinduced electron transfer to 8, but this
chemistry is incompatible with formation of 1 in chemically
synthesized oligonucleotides.26,27 Formation of 1 by irradiating
a phenyl sulfide at 254 nm is also not optimal for working with
DNA.9,28 The dimethoxy substituted aryl sulfide (2, Scheme 1)
provides 1 along with the corresponding carbocation (4) upon
350 nm photolysis.29 The electron-rich aryl sulfide (2) was
incorporated into oligonucleotides by solid-phase synthesis
using phosphoramidite 9, which was prepared in a straightfor-
ward manner from 10 but required acetylation of the C5-
hydroxyl (11) to prevent branching during solid phase
synthesis (Scheme 3).29 Following desilylation, the nucleoside
(12) was carried on to 9 by standard methods. The C5-acetate
group was resistant to the typical mild oligonucleotide
deprotection conditions (K2CO3/CH3OH) but was removed
under more vigorous conditions (concentrated NH4OH/40%
methyl amine; 1:1 by volume; 25 °C, 8 h).30 Control
experiments using 12 showed that the dihydropyrimidine ring
did not fragment under these conditions (data not shown).
Oligonucleotides containing 2 were characterized by ESI-MS.
Duplex DNA containing 2 was destabilized relative to that
containing native thymidine. Dodecamer 14 (Tm = 37.6 0.2
°C) melts almost 10 °C lower than 13 (Tm = 47.2 0.3 °C).31
In contrast to the reactivity of 5-hydroxy-5,6-dihydrothymi-
din-6-yl radical (1) at the 5′-adjacent nucleotide, hydroxide
treatment following aerobic or anaerobic photolysis of 5′-32P-
15 and 5′-32P-16 produced strand scission at the site where 2
5-Hydroxy-5,6-dihydrothymidin-6-yl Radical (1) Does
Not Lead to Direct Strand Breaks or Alkali-Labile
3929
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
was incorporated (19.3−31.3%).31 This indication of abasic site
formation was confirmed via incision by apurinic endonuclease
1 at the nucleotide where 2 was incorporated. The specific
structural identity of the abasic site was determined via
chemical reactivity. Subjecting photolyzed 5′-32P-15 and
5′-32P-16 separately to a series of “fingerprint” reactions ruled
out 2-deoxyribonolactone (L) formation and other reactions
eliminated formation of the C4′-oxidized abasic site (C4-
AP).31,32,34,35 Rather, the reactivity at the original site of 2 was
consistent with AP formation, including the formation of 5′-
cleavage products containing sugar fragments resulting from β-
elimination in addition to phosphate groups at their 3′-
termini.36,37 We ascribe AP formation to the carbocation (4),
which is also produced upon photolysis of the aryl sulfide
(Scheme 4).29 Attribution of the only NaOH labile damage to 4
also indicates that aerobic photolysis of 2 does not produce
diffusible reactive oxygen species (ROS). ROS would have
resulted in direct strand scission and/or NaOH labile lesions at
the position of 2 and at neighboring nucleotides in both
strands. No strand damage is detected in the complementary
strand under any conditions.
midin-6-yl (1) and its respective peroxyl radical (3) are less
reactive than unsubstituted 5 and 7 was probed further using
5′-32P-19, in which uridine is the nucleotide bonded to the 5′-
phosphate of 2. 5,6-Dihydro-2′-deoxyuridin-6-yl radical (5)
yields direct strand breaks by abstracting the C2′-hydrogen
atom from a 5′-adjacent uridine.13,14 The C2′-hydrogen atom
of uridine is considerably weaker (∼86.5 kcal/mol) than any
carbon−hydrogen bond in a 2′-deoxynucleotide and is
accessible in the major groove to the dihydropyrimidine
radicals.39 Despite the more favorable driving force, no
evidence for internucleotidyl hydrogen atom abstraction was
observed upon photolysis of 5′-32P-19 under aerobic or
anaerobic conditions.31 Furthermore, irradiation of the
analogous single stranded oligonucleotides containing 2
(5′-32P-23-27) also failed to produce any direct strand scission
or NaOH labile cleavage at the 5′-adjacent nucleotides in
5′-32P-23 and 5′-32P-24.31
Scheme 4
Slower hydrogen atom abstraction by 1 and 3 than 5 and 7
correlates with the relative reactivity of monomeric 1 and 28
with thiol.29,40 Radical 1 reacted ∼5-times more slowly with β-
mercaptoethanol (BME) than did 28. The differences in rate
constants for reaction with BME between 1 and 28 may be due
to greater steric hindrance in the former. Hydrogen atom
abstraction from a carbon−hydrogen bond is less favorable
thermodynamically than a sulfur−hydrogen bond and likely
proceeds through a later transition state. Consequently, any
correlation between the reactions of alkyl radicals 1 and 28
(which is very similar to 5) and peroxyl radicals 3 and 7 should
result in similar if not greater differences in reactivity with
respect to hydrogen atom abstraction from carbon−hydrogen
bonds. Conformations of the radicals may also contribute to the
differences in reactivity between the actual hydroxyl radical
adduct (1, and peroxyl radical 3) and the respective model
radicals (5 and 7). C5-disubstitution in 3 should favor a
dihydropyrimidine ring conformation in which the methyl
group is pseudo-equatorial and will control the orientation of
the C6-peroxyl radical.41−44 Furthermore, the pseudo-axial
hydroxyl group will perturb base stacking. Depending on the
relative heights of the barriers in the individual steps, the
conformational equilibria may play a role in the reactivity of 3.
Radical 7, which has 2 hydrogen atoms at the C5-position,
should encounter smaller conformational isomerization bar-
riers. A recent computational study suggests that conforma-
tional effects contribute significantly to the barriers for peroxyl
radical reactions in DNA.45
Previous studies on 7 revealed that distance constraints
within helical DNA limit hydrogen atom abstraction from
adjacent 2′-deoxyribonucleotides to the 5′-direction and that
reaction of diastereomeric C6-peroxyl radicals is coupled to
rotation (syn/anti) about the glycosidic bond.15,19 Conse-
quently, we considered the possibility that the configuration
(5R) at C5 in 1 affects the stereoselectivity of O2 trapping and
subsequent reactivity of 3. To probe this, the diastereomer of 2
(20) was incorporated in duplexes (21, 22) of identical
sequence as 15 and 16. The requisite phosphoramidite was
prepared in a similar manner as was 9.31 The 5R-stereo-
chemistry in the precursor (20) was controlled via asymmetric
dihydroxylation of suitably protected thymidine.38 However,
formation of 5S-1 from 20 upon aerobic photolysis of 5′-32P-21
or 5′-32P-22 still did not yield any direct strand breaks or
NaOH labile lesions at the 5′-adjacent nucleotides.31
These data suggested that 3 (and its C5-epimer) is less
reactive than 7. The possibility that 5-hydroxy-5,6-dihydrothy-
3930
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
Tandem Lesion Formation by Peroxyl Radical
Addition to 5′-Adjacent Nucleotides. The peroxyl radical
of 5,6-dihydro-2′-deoxyuridin-6-yl radical (7) yields tandem
lesions by adding to the π-bond of a 5′-adjacent thymidine.15,18
Oxygen radical addition to pyrimidine nucleobases produces
labile lesions, such as thymidine glycol (Tg), which is cleaved
by piperidine. Purine addition yields lesions such as 8-oxo-7,8-
dihydro-2′-deoxyguanosine (8-OxodGuo), which is cleaved
upon incubation with formamido pyrimidine DNA glycosylase
(Fpg) or sequential treatment with Na2IrCl6 and piperidine but
not piperidine by itself.46,47
More definitive product identification was achieved via MS
analysis of photolyzed 29a (Figure 2) and 30.31 Two relatively
The reactivity of peroxyl radical 3 was examined in four
sequences of duplex (15−18) and single stranded (23−26)
DNA in which the identity of the 5′-adjacent nucleotide was
varied (Figure 1). As discussed above neither direct strand
Figure 2. MALDI-TOF MS analysis of photolyzed 29a. (A) Aerobic
and (B) Anaerobic conditions.
abundant products from 29a that were detected under aerobic
and anaerobic conditions corresponded to conversion of 2 to
Tg (29b) or an AP (29e) site. As discussed above, AP is
attributed to carbocation (4) formation. Thymidine glycol was
previously shown to result from formation of the radical (1)
and carbocation (4).29 Another product’s m/z (29c) is
consistent with hydrogen atom transfer to 1 (31), which is
also consistent with its increased intensity following photolysis
under anaerobic conditions without (Figure 2) or with BME (1
mM).31 Aryl thiol produced upon photolysis of 2 is believed to
be the hydrogen atom source in the absence of BME.29 The
peak at m/z = 3630 (29d) that is only observed under aerobic
conditions is proposed to be the tandem lesion (32a) resulting
from addition of 3 to the 5′-adjacent dG. The dG is ultimately
transformed into 8-OxodGuo, and the peroxyl radical fragments
to produce the formamide lesion. Tandem lesion 32a was
previously observed when DNA is exposed to •OH under
aerobic conditions.21−23 Although gel electrophoresis experi-
ments (Figure 1) indicate that tandem lesions other than those
containing 8-OxodGuo are formed, none were detected by
MALDI-TOF MS (Figure 2). We also identified a tandem
lesion from 30 by LC/ESI-MS.31 An ion that corresponds to
Figure 1. DNA strand lability at nucleotides 5′-adjacent to 2 following
aerobic photolysis. (A) Double-stranded substrates (5′-32P-15-18). (B)
Single-stranded substrates (5′-32P-23-26).
breaks or abasic lesions are produced at this position, but
varying amounts of nucleobase damage were detected. In
addition, damage is undetectable in the complementary strand.
Labile damage at a 5′-adjacent dG (dG15) was more than 5-
times greater than at the other three native nucleotides.
Furthermore, cleavage at dG15 in photolyzed 5′-32P-16 was
more than 3-fold greater following treatment with Fpg or
Na2IrCl6 followed by piperidine than with piperidine alone,
indicating that 8-OxodGuo is not the only lesion formed.
Incision also increased ∼3-fold upon Na2IrCl6/piperidine
treatment in the single stranded substrate (24). The formation
of all labile lesions was dependent on O2, consistent with the
involvement of 3.31
3931
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
the formation of tandem lesion containing formamide and
HOMedU (32b) was detected.
Previously, the reactivity of 7 had only been examined in
sequences containing a 5′-thymidine.15,18 5,6-Dihydro-2′-
deoxyuridin-6-yl (5) was generated in the comparable
sequences containing 5′-adjacent dG or T (33−36) to probe
the generality of the preference for reactivity with dG. Overall,
the level of alkali-labile damage in DNA was greater when 7 was
generated than was 3. However, the same strong preference for
damage at dG15 compared to T15 was observed (Figure 3).
Figure 4. Effect of BME on tandem lesion formation in duplex DNA.
5′-32P-16 and 5′-32P-34 were photolyzed.
the amount of cleavage product following treatment of the
photolysate with excess Fpg or Na2IrCl6/piperidine. (The ratios
of rate constants were independent of the postphotolysis
treatment.) Subtracting the amount of strand scission at dG15 in
the presence of thiol at a given concentration from that in the
absence of thiol provided the amount of thiol trapping. These
experiments were carried out at sufficiently low thiol levels so
that BME trapping of 1 and 5 did not compete with O2. By
assuming that ktrap of the peroxyl radicals in DNA by BME is 2
× 102 M−1 s−1,48 we estimate that 3 (kcleave = 7.3 0.9 × 10−2
s−1) reacts with the 5′-adjacent dG15 approximately half as fast
as does 7 (kcleave = 12.2 1.5 × 10−2 s−1).
The thiol trapping data are consistent with the observations
noted above regarding the relative abilities of 3 and 7 to
abstract hydrogen atoms from the 2′-deoxyribose portions of
DNA and react with adjacent nucleobases (Figures 1 and 3).
The difference likely represents a maximum that will be
reduced by any differences in reactivity between BME and the
peroxyl radicals. Comparing kcleave for 7 with that reported
previously for this peroxyl radical’s reaction with a 5′-T also
reinforces the qualitative comparisons of peroxyl radical
reactivity showing that dG is more readily damaged than T
(Figures 1 and 3).18 The competitive kinetic experiments
indicate that 7 reacts with a 5′-dG ∼28-times faster than with a
5′-T.
Figure 3. DNA strand lability at nucleotides 5′-adjacent to 6 following
aerobic photolysis. (A) Double-stranded substrates (5′-32P-33, 34).
(B) Single-stranded substrates (5′-32P-35, 36).
Distal Oxidation via 5,6-Dihydropyrimidine Peroxyl
Radical Formation. DNA oxidation occurs over long
distances via electron transfer (often referred to as hole
transfer/migration).49−53 The damage ultimately settles prefer-
entially at 2′-deoxyguanosine because it is the most readily
oxidized nucleotide.54 18O-Labeling studies indicate that
pyrimidine peroxyl radicals add into a guanine ring, but it is
not known if the subsequently formed radical is capable of
initiating hole migration.22 More recently, electron transfer
between dG and pyrimidine peroxyl radicals has been proposed
to account for approximately one-half of the 8-OxodGuo
produced by •OH, despite electron transfer from dG to a
peroxyl radical being thermodynamically uphill by ∼0.23 V.24,55
We combined our ability to independently generate 3 and 7
with the wealth of information available regarding hole transfer
in DNA to examine the proposal that a pyrimidine peroxyl
radical can initiate electron transfer by oxidizing dG. 5′-dGGG
is the most readily oxidized trinucleotide sequence, and it is
frequently used as a trap for holes in DNA.7,56−59 Depending
upon the flanking sequence, either the 5′-terminal dG or the
central dG within 5′-dGGG is most readily oxidized as a result
Damage at dG was preferred by almost 3-fold over T in single
stranded oligonucleotides (35, 36) and almost 4-fold in duplex
substrates 33 and 34 in which peroxyl radical 7 was produced.
To probe whether the higher tandem lesion yields from 6 are
due to faster reaction of 7 compared to 3, we carried out
competition experiments using BME (Figure 4, eq 1).
k
trap[3 or 7][BME]
cleave[3 or 7]
[trapped]
[cleaved]
=
k
(1)
Assuming that the reactivity−selectivity principle is applicable,
the difference between reaction rate constants of the peroxyl
radicals with BME will be small compared to 5′-adjacent purine
addition. Hence, comparing the slopes of lines obtained from
plotting the ratio of thiol trapping to alkali-labile product at the
5′-adjacent nucleotide versus [BME] provides an estimate of
the lower limit of relative rate constants for the reaction of
peroxyl radicals 3 and 7 with dG15 in 16 and 34, respectively.
The amount of alkali-labile product was obtained directly from
3932
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
damage pattern expected for an electron transfer process.31
Moreover, the same trend was observed from alkaline cleavage
in the 5′-dGGG sequence of 38b. The damage detected at dG13
was 2−3 times greater than at dG12 and dG11, again
inconsistent with electron transfer. As an aside, the cleavage
yields at the dG’s in each substrate were different when treated
with piperidine or Na2IrCl6/piperidine, suggesting that 8-
OxodGuo was not the only lesion formed at these
nucleotides.46,47
Final tests for electron transfer were carried out using 39a
and 39b. Holes migrate from one strand to another in duplex
DNA, whereas an addition mechanism will be more limited by
conformational constraints imposed by the biopolymer.7 While
alkaline damage was detected at dG15 in 39a and 39b, no strand
damage was detected at dG47−49 in either substrate. In addition
to providing additional evidence against dG oxidation by
electron transfer following pyrimidine peroxyl radical (3, 7)
formation, the absence of strand damage at dG47−49 in 39a and
39b provides additional evidence against the involvement of a
diffusible reactive oxygen species.31
Having ruled out electron transfer and diffusible reactive
species, an alternative explanation for distal oxidation (dG13) in
38a,b was sought. One possibility involves addition of the
initially generated peroxyl radical (3, 7) to the distal purine.
Reaction at dG13 requires the duplex to adopt a conformation
that enables the peroxyl radical to approach the purine 3
nucleotides removed (Scheme 5). UV melting experiments
reveal that the dihydropyrimidine photochemical precursors
destabilize the duplexes, and computations on related
molecules suggest that the peroxyl radicals are likely to as
well.41,42 However, we are unaware of a reaction between two
nucleotides this far away from one another in duplex DNA.
Alternatively, the peroxyl radical initially formed could react
with 5′-adjacent dG15 and a reactive intermediate on the purine
that results from this process could act as a shuttle and transfer
damage to dG13 (Scheme 5). Reaction between guanyl radicals
in single stranded and duplex DNA with a nucleotide two
positions away has been observed.61,62 Recently, such lesions
were even detected in irradiated HeLa cells.63
of hole transfer.60 A series of duplexes containing a 5′-dGGG
sequence and either 2 or 6 were prepared to probe for electron
transfer (37−39). Curiously, strand damage in 37 was less than
in the comparable duplex (16) containing a single dG adjacent
to 2 (Figure 1A). Moreover, strand damage at dG15 (1.5
0.4%) of 37, which is the 3′-terminal nucleotide in the 5′-
dGGG sequence was greater than at dG14 (0.5 0.1%) and
dG13 (none detected).31 This is the opposite selectivity for
damage expected if electron transfer is involved.
Although experiments with 37 suggested that 3 was unable to
oxidize a 5′-dG by outer sphere electron transfer, we considered
the possibility that addition of peroxyl radical 3 to an adjacent
dG produces an intermediate(s) that initiates electron transfer.
Consequently, duplexes (38a and 38b) containing a 5′-dGGG
sequence separated from the 5′-adjacent dG by one base pair
were prepared. Overall alkali-labile damage was 4−5 times
greater in 38b (Figure 5B) than in 38a. This is consistent with
These possibilities were explored by comparing the damage
induced in 38b with that in 40 and 41. Replacing dG15 in 38b
(Figure 5B, alkali-labile cleavage at dG13: 9.7
2.4%) with
thymidine resulted in a large reduction in damage at dG13 in 40
(Figure 6, alkali-labile cleavage at dG13: 3.8 1.0%). This is
consistent with generation of a reactive species at dG15 of 38b
that acts as a shuttle to transfer damage two nucleotides further
in the 5′-direction to dG13. The effect of distance between dG15
and the 3′-terminal dG in the 5′-dGGG sequence was
examined by adding a thymidine (41). Greater damage is
observed at T13 (4.1 0.2%, Figure 6) than at a T in any other
duplex. Furthermore, alkali-labile damage at dG12 is the same
Figure 5. DNA strand lability following aerobic photolysis. (A) 5′-32P-
38a. (B) 5′-32P-38b.
the greater nucleobase radical yield from 6 than from 2 and the
higher reactivity described above for 7 compared to 3.29 Per
above, no damage was detected under anaerobic conditions.
Overall strand damage was greater in 38a (Figure 5A) than in
37, and the 3′-terminal dG of the 5′-dGGG sequence was most
susceptible to either piperidine or Na2IrCl6/piperidine cleavage.
Preferential damage at dG13, the 3′-terminal dG in 5′-dGGG,
over dG12 and dG11 (not detected), is inconsistent with the
within experimental error in 38b (3.7
1.5%), 40 (2.7
0.8%), and 41 (2.2 0.5%, Figure 6). These observations are
also consistent with formation of a reactive intermediate at
dG15 capable of reacting two nucleotides away.
3933
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
Scheme 5
double that in dG12 in 41 (Figure 6). These observations
further illustrate how greater conformational mobility in single
stranded substrates facilitates reactivity toward the most readily
oxidized nucleotides, which are the dGs.
Overall, these experiments indicate that a 5′-adjacent dG can
react with a pyrimidine peroxyl radical and help transfer
damage to a more distal nucleotide in duplex DNA. However,
these experiments do not rule out a small contribution from
direct reaction between a pyrimidine peroxyl radical and a
nucleobase up to three nucleotides away, as observed for single
stranded 42 (Figure 7, Scheme 5). These reactions will produce
complex, multiply damaged lesions, which are increasingly
common in ionizing radiation and believed to be biologically
significant.16,64 Attempted characterization of the complex
lesions by LC/MS was unsuccessful. This may be due to the
formation of multiple combinations of damaged nucleotides at
up to three positions in one oligonucleotide.
Figure 6. DNA strand lability following aerobic photolysis of 5′-32P-40
and 5′-32P-41. Cleavage was induced with piperidine following
treatment with Na2IrCl6.
In contrast, the presence of dG15 between 7 and dG13 has no
apparent effect in the more conformationally mobile single
stranded oligonucleotides (Figure 7). The alkali-labile damage
at dG13 in 42 and 44 is within experimental error of one
another. Furthermore, the alkali-labile damage yield at T13 in 43
is not any greater than at any thymidine in any of the other
substrates examined, while the yield of damage at dG12 in 43 is
CONCLUSIONS
■
These experiments reveal that the major hydroxyl radical
adduct of thymidine (1) does not produce detectable levels of
direct strand breaks or alkali-labile clustered lesions. The
respective peroxyl radical (3) of 5-hydroxy-5,6-dihydrothymi-
din-6-yl radical (1) is less reactive than unsubstituted analogue
7 and does not yield measurable levels of hydrogen atom
abstraction products. The source of the lower reactivity of 3
compared to 7 is uncertain but sterics could play a role.
Disubstitution at C5 of the dihydropyrimidine destabilizes base
stacking and may increase the energy of conformations
necessary to achieve the internucleotide reactions dis-
cussed.41,42,65 Recent computational studies on dinucleotide
reactions involving peroxyl radicals affirm the importance of the
approach trajectory.45
Figure 7. DNA strand lability following aerobic photolysis of 5′-32P-
42−44. Cleavage was induced with piperidine following treatment
with Na2IrCl6.
3934
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
(16) Sage, E.; Harrison, L. Mutat. Res. 2011, 711, 123.
(17) Kozmin, S. G.; Sedletska, Y.; Reynaud-Angelin, A.; Gasparutto,
D.; Sage, E. Nucleic Acids Res. 2009, 37, 1767.
(18) Hong, I. S.; Carter, K. N.; Sato, K.; Greenberg, M. M. J. Am.
Chem. Soc. 2007, 129, 4089.
Despite steric differences, 3 and 7 react with adjacent and
nearby nucleobases, producing potentially mutagenic clustered
lesions. The clustered lesions result from covalent reactions of
the peroxyl radicals with neighboring nucleotides, preferably
dG. The initial species produced from reaction at a 5′-adjacent
dG may act as a shuttle by transferring damage to more distal
nucleotides, creating clustered lesions that consist of three
oxidatively modified nucleotides. There is no evidence for
peroxyl radicals 3 or 7 initiating electron transfer in duplex
DNA by oxidizing dG. Biochemically deleterious lesions
consisting of multiply damaged nucleotides are produced by
ionizing radiation, and these experiments suggest that they may
result from a single event between hydroxyl radical and DNA.
(19) Hong, I. S.; Carter, K. N.; Greenberg, M. M. J. Org. Chem. 2004,
69, 6974.
(20) Patrzyc, H. B.; Dawidzik, J. B.; Budzinski, E. E.; Freund, H. G.;
Wilton, J. H.; Box, H. C. Radiat. Res. 2012, 178, 538.
(21) Box, H. C.; Patrzyc, H. B.; Dawidzik, J. B.; Wallace, J. C.;
Freund, H. G.; Iijima, H.; Budzinski, E. E. Radiat. Res. 2000, 153, 442.
(22) Douki, T.; Riviere, J.; Cadet, J. Chem. Res. Toxicol. 2002, 15, 445.
(23) Bourdat, A.-G.; Douki, T.; Frelon, S.; Gasparutto, D.; Cadet, J. J.
Am. Chem. Soc. 2000, 122, 4549.
(24) Bergeron, F.; Auvre, F.; Radicella, J. P.; Ravanat, J.-L. Proc. Natl.
́
Acad. Sci. U.S.A. 2010, 107, 5528.
(25) Carter, K. N.; Greenberg, M. M. J. Org. Chem. 2003, 68, 4275.
(26) Barvian, M. R.; Greenberg, M. M. Tetrahedron Lett. 1992, 33,
6057.
(27) Barvian, M. R.; Barkley, R. M.; Greenberg, M. M. J. Am. Chem.
Soc. 1995, 117, 4894.
(28) Zhang, Q.; Wang, Y. J. Am. Chem. Soc. 2004, 126, 13287.
(29) San Pedro, J. M. N.; Greenberg, M. M. Org. Lett. 2012, 14, 2866.
(30) Reddy, M. P.; Hanna, N. B.; Faroqui, F. Tetrahedron Lett. 1994,
35, 4311.
(31) See Supporting Information.
(32) San Pedro, J. M. N.; Beerman, T. A.; Greenberg, M. M. Bioorg.
Med. Chem. 2012, 20, 4744.
ASSOCIATED CONTENT
* Supporting Information
■
S
Procedures for all experiments, MS of photolyzed 29a and 30,
UV melting curves, representative autoradiograms, NMR
spectra of new compounds, and mass spectra of oligonucleo-
tides containing nonnative nucleotides. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
mgreenberg@jhu.edu
■
Notes
́
(33) Pitie, M.; Pratviel, G. Chem. Rev. 2010, 110, 1018.
(34) Hwang, J.-T.; Tallman, K. A.; Greenberg, M. M. Nucleic Acids
Res. 1999, 27, 3805.
The authors declare no competing financial interest.
(35) Zheng, Y.; Sheppard, T. L. Chem. Res. Toxicol. 2004, 17, 197.
(36) Sugiyama, H.; Kawabata, H.; Fujiwara, T.; Dannoue, Y.; Saito, I.
J. Am. Chem. Soc. 1990, 112, 5252.
(37) Sugiyama, H.; Fujiwara, T.; Ura, A.; Tashiro, T.; Yamamoto, K.;
Kawanishi, S.; Saito, I. Chem. Res. Toxicol. 1994, 7, 673.
(38) Barvian, M. R.; Greenberg, M. M. J. Org. Chem. 1993, 58, 6151.
(39) Li, M.-J.; Liu, L.; Wei, K.; Fu, Y.; Guo, Q.-X. J. Phys. Chem. B
2006, 110, 13582.
ACKNOWLEDGMENTS
■
We are grateful for generous financial support from the
National Institute of General Medical Sciences (GM-054996).
J.M.N.S.P. is grateful for the Martin and Mary Kilpatrick
Fellowship from Johns Hopkins University. We thank Dr. Phil
Mortimer and Dr. Marisa L. Taverna Porro for their assistance
with the LC/ESI-MS experiments.
(40) Newman, C. A.; Resendiz, M. J. E.; Sczepanski, J. T.; Greenberg,
M. M. J. Org. Chem. 2009, 74, 7007.
(41) Miaskiewicz, K.; Miller, J.; Ornstein, R.; Osman, R. Biopolymers
1995, 35, 113.
(42) Miaskiewicz, K.; Miller, J.; Osman, R. Biochim. Biophys. Acta
REFERENCES
■
(1) Hirayama, R.; Ito, A.; Noguchi, M.; Matsumoto, Y.; Uzawa, A.;
Kobashi, G.; Okayasu, R.; Furusawa, Y. Radiat. Res. 2013, 180, 514.
(2) von Sonntag, C. Free-Radical-Induced DNA Damage and Its
Repair; Springer-Verlag: Berlin, 2006.
(3) Pogozelski, W. K.; McNeese, T. J.; Tullius, T. D. J. Am. Chem.
Soc. 1995, 117, 6428.
(4) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9738.
(5) Greenberg, M. M. Org. Biomol. Chem. 2007, 5, 18.
(6) Deeble, D. J.; Schulz, D.; Von Sonntag, C. Int. J. Radiat. Biol.
1986, 49, 915.
1994, 1218, 283.
(43) Resendiz, M. J. E.; Schon, A.; Freire, E.; Greenberg, M. M. J. Am.
̈
Chem. Soc. 2012, 134, 12478.
(44) San Pedro, J. M. N.; Greenberg, M. M. ChemBioChem 2013, 14,
1590.
(45) Dupont, C.; Patel, C.; Ravanat, J. L.; Dumont, E. Org. Biomol.
Chem. 2013, 11, 3038.
(46) Muller, J. G.; Duarte, V.; Hickerson, R. P.; Burrows, C. J. Nucleic
Acids Res. 1998, 26, 2247.
(47) Cullis, P. M.; Malone, M. E.; Merson-Davies, L. A. J. Am. Chem.
Soc. 1996, 118, 2775.
(48) Hildenbrand, K.; Schulte-Frohlinde, D. Int. J. Radiat. Biol. 1997,
71, 377.
(7) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc.
1998, 120, 12950.
(8) Amato, N. J.; Bryant-Friedrich, A. C. ChemBioChem 2013, 14,
187.
(9) Zhang, Q.; Wang, Y. Chem. Res. Toxicol. 2005, 18, 1897.
(10) Hildenbrand, K.; Schulte-Frohlinde, D. Int. J. Radiat. Biol. 1989,
55, 725.
(49) Slinker, J. D.; Muren, N. B.; Renfrew, S. E.; Barton, J. K. Nat.
Chem. 2011, 3, 228.
(50) Genereux, J. C.; Barton, J. K. Chem. Rev. 2010, 110, 1642.
(51) Joy, A.; Schuster, G. B. Chem. Commun. 2005, 2778.
(52) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res.
2001, 34, 159.
(11) Milligan, J. R.; Aguilera, J. A.; Nguyen, T.-T. D. Radiat. Res.
1999, 151, 334.
(12) Resendiz, M. J. E.; Pottiboyina, V.; Sevilla, M. D.; Greenberg, M.
M. J. Am. Chem. Soc. 2012, 134, 3917.
(53) Giese, B. Acc. Chem. Res. 2000, 33, 631.
(54) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617.
(55) Jovanovic, S. V.; Jankovic, I.; Josimovic, L. J. Am. Chem. Soc.
1992, 114, 9018.
(13) Jacobs, A. C.; Resendiz, M. J. E.; Greenberg, M. M. J. Am. Chem.
Soc. 2011, 133, 5152.
(14) Jacobs, A. C.; Resendiz, M. J. E.; Greenberg, M. M. J. Am. Chem.
Soc. 2010, 132, 3668.
(56) Saito, I.; Nakanura, T.; Nakatani, K.; Yoshioka, Y.; Yamaguchi,
(15) Carter, K. N.; Greenberg, M. M. J. Am. Chem. Soc. 2003, 125,
13376.
K.; Sugiyama, H. J. Am. Chem. Soc. 1998, 120, 12686.
3935
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936

Journal of the American Chemical Society
Article
(57) Davis, W. B.; Bjorklund, C. C.; Deline, M. Biochemistry 2012, 51,
3129.
(58) Liu, Y.; Liu, Z.; Geacintov, N. E.; Shafirovich, V. Phys. Chem.
Chem. Phys. 2012, 14, 7400.
(59) Nakatani, K.; Dohno, C.; Saito, I. J. Am. Chem. Soc. 2000, 122,
5893.
(60) Yoshioka, Y.; Kitagawa, Y.; Takano, Y.; Yamaguchi, K.;
Nakamura, T.; Saito, I. J. Am. Chem. Soc. 1999, 121, 8712.
(61) Crean, C.; Uvaydov, Y.; Geacintov, N. E.; Shafirovich, V. Nucleic
Acids Res. 2008, 36, 742.
(62) Yun, B. H.; Geacintov, N. E.; Shafirovich, V. Chem. Res. Toxicol.
2011, 24, 1144.
(63) Madugundu, G. S.; Wagner, J. R.; Cadet, J.; Kropachev, K.; Yun,
B. H.; Geacintov, N. E.; Shafirovich, V. Chem. Res. Toxicol. 2013, 26,
1031.
(64) Eccles, L. J.; Lomax, M. E.; O’Neill, P. Nucleic Acids Res. 2010,
38, 1123.
(65) Miaskiewicz, K.; Osman, R. J. Am. Chem. Soc. 1994, 116, 232.
3936
dx.doi.org/10.1021/ja412562p | J. Am. Chem. Soc. 2014, 136, 3928−3936