Direct strand scission in double stranded RNA via a C5-pyrimidine radical

Article abstract of DOI:10.1021/ja300044e

Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-dihydrouridin-5- yl radical (1), the formal product of hydrogen atom addition and a model for hydroxyl radical addition, was independently generated from a ketone precursor via Norrish Type I photocleavage in single and double stranded RNA. Radical 1 produces direct strand breaks at the 5′-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O₂. The dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3′-phosphate following C2′-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical yields RNA fragments containing 3′-phosphate or 3′-deoxy-2′-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl radical (1) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.

Full text of DOI:10.1021/ja300044e

Article
pubs.acs.org/JACS
Direct Strand Scission in Double Stranded RNA via
a C5-Pyrimidine Radical
Marino J. E. Resendiz,† Venkata Pottiboyina,‡ Michael D. Sevilla,‡ and Marc M. Greenberg*,†
†Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
‡Department of Chemistry, Oakland University, Rochester, Michigan 48309, United States
S
* Supporting Information
ABSTRACT: Nucleobase radicals are the major family of reactive inter-
mediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-
dihydrouridin-5-yl radical (1), the formal product of hydrogen atom addition
and a model for hydroxyl radical addition, was independently generated from a
ketone precursor via Norrish Type I photocleavage in single and double
stranded RNA. Radical 1 produces direct strand breaks at the 5′-adjacent
nucleotide and only minor amounts of strand scission are observed at the
initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O2. The
dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic
conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical
reactions. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission
occurs via β-elimination of the 3′-phosphate following C2′-hydrogen atom abstraction by 1. The subsequently formed
olefin cation radical yields RNA fragments containing 3′-phosphate or 3′-deoxy-2′-ketonucleotide termini from competing
deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by
reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl
radical (1) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies
using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.
radical shown to form direct strand breaks in RNA by abstract-
ing hydrogen atoms from the ribose backbone.9,10 However,
there are significant differences between the reactivity of 1 and 5,
6-dihydrouridin-6-yl (2), which are delineated below.
INTRODUCTION
■
RNA cleavage is an important tool for analyzing the structure
and folding kinetics of this biopolymer.1 The hydroxyl radical
(OH•), produced by Fe•EDTA or pulse radiolysis, is the most
commonly used reagent for this purpose.2,3 RNA damage induced
by reactive oxygen species is also increasingly recognized to
play a role in diseases and aging. Extensive research by radia-
tion scientists has established that OH• and hydrogen atoms
preferentially add to the π-bonds of RNA nucleobases.4,5 The
nucleobase radicals also arise from hydration of the cation
radicals formed via direct ionization of nucleic acids. Hydrogen
atom abstraction by OH• from the ribose rings has been
estimated to account for less than 10% of the reactions with
polynucleotides. Despite this, RNA cleavage by OH• under
aerobic conditions is typically ascribed to hydrogen abstraction
by this radical from the C4′- and C5′-positions of the ribose
backbone. The high reactivity of OH• suggests that it will
exhibit little chemical selectivity. Preferential abstraction of the
C4′- and C5′-hydrogen atoms is attributed to their high ac-
cessibility to diffusible species.6 The resulting C4′- and C5′-
radicals yield direct strand breaks by mechanisms that are
partially understood.7,8 In contrast, strand scission from nucleo-
base radicals requires subsequent hydrogen atom abstraction
from a ribose ring. Herein we describe the reactivity of the 5,6-
dihydrouridin-5-yl radical (1) and show that it produces direct
strand breaks by abstracting a hydrogen atom from the ribose
backbone. 5,6-Dihydrouridin-5-yl (1) is the second nucleobase
Hydrogen atoms and OH• preferentially add to the more
electron rich C5-carbon of the uracil double bond. However,
formation of the regioisomeric C5-radicals (1, 3) can account
for up to 40% of the addition reactions.5 The C5-radicals are
also preferentially formed upon hydration of cation radicals
produced via direct ionization.11 Various proposals concerning
the subsequent reactivity of these nucleobase radicals have been
put forth. In polynucleotides the nucleobase radicals have been
proposed to yield strand breaks by abstracting hydrogen atoms
from the C4′- and (in RNA) C2′-positions of the sugar
backbone.12−15 The cation radicals have also been proposed to
yield strand breaks by abstracting ribose hydrogen atoms from
Received: January 3, 2012
Published: February 15, 2012
© 2012 American Chemical Society
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the C4′- and C2′-positions.16,17 In some instances deciphering
the intermediate(s) responsible for strand scission resulting
from nucleobase intermediates has been difficult due to hy-
dration of the cation radicals as well as the reverse process,
dehydration of hydroxyl radical adducts.14 Determining the
nucleotide from which the nucleobase radical abstracts hy-
drogen atom(s) was also difficult in these experiments because
the initially formed reactive intermediates were generated ran-
domly within the biopolymers. Independent generation of reac-
tive intermediates within nucleic acids greatly simplifies eluci-
dating the chemistry of nucleic acid damage and has provided
significant insight into DNA damage and repair.18−22 Recently,
we applied this approach to study RNA damage and demon-
strated that 5,6-dihydrouridin-6-yl (2) (generated via Norrish
Type I photocleavage) produces direct strand breaks by ab-
stracting hydrogen atom(s) from its own ribose ring (intra-
nucleotidyl) and the C2′-hydrogen atom from the 5′-adjacent
nucleotide (internucleotidyl) (Scheme 1).9,10,23 The C2′-radical
a
Scheme 2.
a
Key: (a) LDA, PivCI; (b) TFA, H2O; (c) DMTCI; (d) TBDMSCI,
AgNO3; (e) Phosphitylation.
Scheme 1
phase synthesis. Oligonucleotides containing 6 were depro-
tected using K2CO3 in methanol, desilylated (Et3N•3HF,
N-methylpyrrolidinone),32 and purified by denaturing poly-
acrylamide gel electrophoresis.
The 5′-dimethoxytritylated intermediate (10) was used in
monomer photochemical studies to establish the integrity of
the radical generation process. High mass balances (Table 1)
Table 1. Product Yields from the Photochemical Generation
of 5,6-Dihydrouridin-5-yl (1) from 10
a
Yield (%)
b
Conditions
10
12
13
Mass Balance
Anaerobic
Aerobic
57.3 2.4
62.5 2.9
27.7 2.6
−
85.0 2.3
91.7 4.2
−
29.2 1.4
a
Yields are the average of 2 independent experiments each carried out
b
in triplicate. Starting [10] = 100 μM, [BME] = 5 mM.
undergoes β-elimination to yield a strand break in which the 5′-RNA
fragment contains a cation radical (5, Scheme 1) at its 3′-terminus.
were obtained when aqueous acetonitrile solutions (1:1 by
volume) of 10 (100 μM) were irradiated in the cavity of a
Rayonet photoreactor using lamps that emit with maximum
intensity at 350 nm. Dimethoxytritylated dihydrouridine (12)
and the correspondingly protected 5-hydroxy-5,6-dihydrour-
idine (13) were the only products observed under anaerobic
and aerobic conditions in the presence of β-mercaptoethanol
(BME, 5 mM) (Table 1). Photoconversion of 10 is qualitatively
less efficient than generation of 2 from the respective tert-butyl
ketone. This is due at least in part to photoenolization, which
RESULTS AND DISCUSSION
■
Precursor Synthesis and Photochemical Generation of
the 5,6-Dihydrouridin-5-yl Radical (1). 5,6-Dihydrouridin-5-
yl (1) is the formal C6-hydrogen atom adduct of uri-
dine and a model of the respective OH• adduct (3). It was
generated in this study from 6 (eq 1) via Norrish Type I
2
was detected by H incorporation in recovered 14 that was
photolyzed in D2O and analyzed by MALDI-TOF MS.33
Dihydrouridine 12 was also the sole product detected when 10
was photolyzed under anaerobic conditions in the presence of
high concentrations of either BME or 2-propanol. Based upon
unrecovered starting material, the yield of 12 ranged from 65 to
98% when the BME concentration was varied between 0.5 and
2.5 M and from 50 to 65% when 2-propanol (1.5−10 M) was
used as a hydrogen atom donor. Mass balances in these reac-
tions ranged from 76 to 80% in the presence of BME and 75−
95% with 2-propanol. Importantly, photoreduction product 15
was not detected in the presence of even the highest concen-
trations of either hydrogen atom donor. This suggests that the
excited state of ketone 6 would not abstract hydrogen atoms
from the ribose backbone.
photocleavage, a photochemical reaction that has been used
to generate a number of nucleic acid radicals, including
the regioisomeric 5,6-dihydrouridin-6-yl radical (2).23−30 The
ketone (8) was synthesized by acylating protected dihydrour-
idine (7, Scheme 2), as previously described.31 A mixture of
diastereomers of 8 was transformed into phosphoramidite 11
by standard methods via the free nucleoside (9). Phosphor-
amidite 11 was incorporated into oligoribonucleotides via solid
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2-fold greater than that of 2.9,10 The reactivity of the two
radicals under aerobic conditions is also very similar. A complex
mixture of direct strand scission products is detected in low
yield from 1 produced in single and double stranded RNA
under aerobic conditions and was not investigated further.
Strand scission from the 5,6-dihydrouridin-5-yl radical (1) is
distributed between the 5′-adjacent nucleotide (internucleotidyl)
and nucleotide at which 1 is generated (intranucleotidyl). The
preference for internucleotidyl strand scission in duplex RNA is
slightly greater from 1 than from the 5,6-dihydrouridin-6-yl
radical (2).9,10 More than 90% of the strand breaks in
double stranded RNA from 1 (compared to 85% from 2) are
formed following reaction with the 5′-adjacent nucleotide re-
gardless of whether the radical is flanked by U or A. Although
the mechanism for intranucleotidyl strand scission is unknown
for either radical, conformational constraints suggest that the
5,6-dihydrouridin-5-yl radical (1) would be unable to abstract
hydrogen atoms from the C2′- or C3′-position. Examination of
molecular models suggests that reaction at the C5′-position is
more likely, but there is no evidence to support this pathway.
Also it is possible that the small amounts of intranucleotidyl
strand scission in photolyzed oligonucleotides containing 6 are
an artifact. A greater difference in nucleotide selectivity between
the nucleobase radicals is observed in single stranded sub-
strates. Whereas as much as 82% of strand scission from the
5,6-dihydrouridin-6-yl radical (2) in single stranded substrates
occurs at the nucleotide in which the radical is originally
generated, less than 50% of the cleavage from 1 is a result of
intranucleotidyl reaction.9,10 Unlike 2 from which the absolute
amount of intranucleotidyl strand scission is greater in single
stranded RNA than double stranded, the absolute yield of
intranucleotidyl strand scission from the 5,6-dihydrouridin-5-yl
radical (1) is independent of the secondary structure of the
RNA in which it is produced. This too is consistent with the
possibility that cleavage at the nucleotide in which 1 is gen-
erated may be an artifact.
Direct Strand Scission from the 5,6-Dihydrouridin-5-
yl Radical (1). Strand scission from 1 was examined in
oligonucleotides in which the radical was flanked by uridine
(16, 18) or adenosine (17, 19, Table 2). Overall, direct strand
Table 2. Direct Strand Scission under Anaerobic Conditions
from 5,6-Dihydrouridin-5-yl (1)
Characterization of the Strand Scission Products.
Products were characterized using enzymatic end group analysis
and by MALDI-TOF MS.10,33 Formation of the 5,6-dihydro-
uridin-5-yl radical (1) under anaerobic conditions in single or
double stranded RNA produced differing distributions of the
same set of products that were separable by 20% denaturing
PAGE. These products were observed regardless of whether the
radical precursor was flanked by U or A. Calf alkaline phos-
phatase treatment of photolyzed 3′-32P-16 confirmed that 5′-
phosphates (22, 23) were the exclusive products at the intra-
nucleotidyl and 5′-adjacent nucleotide cleavage sites.33 Poly-
nucleotide kinase treatment of 5′-32P-16 that was photolyzed
under anaerobic conditions revealed that two of the products
contained 3′-phosphates. The migrations of these products
were consistent with 3′-phosphorylated products resulting from
inter- (24) and intranucleotidyl cleavage (25a,b). A third pro-
duct that migrated more slowly than either 3′-phosphate did
not react with kinase (26a,b). The migration of this product
through a 20% gel was altered by treatment with NaBH4,
although the effect of the reducing agent differed depending
upon whether 1 was flanked by U or A (eq 2).33 Treatment of
the product formed from the 5′-U (5′-32P-16) flanking sequence
produced a slower migrating product (28a), whereas photolysis
of 5′-32P-17 containing a 5′-A yielded a product that upon
reduction moved faster through the gel (28b). Regardless, reac-
tion with NaBH4 suggested that formation of the 5,6-dihydro-
uridin-5-yl radical (1) in either sequence under anaerobic
a
Absolute yield (%)
b
c
d
c
Substrate
Inter.
Intra.
Total
% Inter.
ds U6U (16)
ss U6U (18)
ds A6A (17)
ss A6A (19)
31.9 3.0
3.4 0.6
26.1 3.8
5.4 1.4
2.0 0.7
3.2 0.2
2.4 1.6
2.4 0.6
33.9 3.0
6.9 1.3
28.6 3.5
8.0 1.5
94.1 1.9
52.1 13.5
91.3 6.0
68.0 4.5
a
Yields are the average of at least three separate experiments each
carried out in triplicate. The substrate is designated by its secondary
structure (ss, ds). Inter. corresponds to strand scission at the 5′-
adjacent nucleotide from 6. Intra. corresponds to strand scission at
b
c
d
the nucleotide where 1 is generated.
scission from the 5,6-dihydrouridin-5-yl radical (1) was most
efficient in double stranded substrates under anaerobic con-
ditions. The 5,6-dihydrouridin-6-yl radical (2) also yielded the
highest strand scission yields in duplexes under anaerobic
conditions.9,10 Direct strand scission in double stranded RNA
(17) was 3.6
0.8 times greater than in a single stranded
substrate (19) when 1 was flanked by adenosine (Table 2). The
average preference for cleavage in duplex RNA (16) was greater
when the 5,6-dihydrouridin-5-yl radical (1) was flanked by
uridine (4.9 1.0) but within experimental error of that ob-
served when 1 was flanked by adenosine. The preference for
strand scission in double stranded compared to single stranded
RNA by the 5,6-dihydrouridin-5-yl radical (1) is approximately
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resulting from intranucleotidyl cleavage (27a,b) was not
observed.
Inferential characterization of 26a was validated by MALDI-
TOF MS analysis of 16 photolyzed at pH 3.8 (Figure 1). In
addition to 26a, the 3′-phosphorylated product resulting from
internucleotidyl strand scission (24) was also observed but not
the minor product resulting from intranucleotidyl strand scis-
sion (25a). Another significant product detected in the MS
spectrum is ascribed to the 3′-fragment formed upon scission at
the 5′-adjacent nucleotide (23). The ion of this oligonucleotide
corresponds to a molecule containing a 5′-phosphorylated
dihydrouridine at its 5′-terminus (23), which is what one would
expect if strand scission was due to hydrogen atom abstraction
by 1 from the 5′-adjacent nucleotide.
The ratio of 24:26a,b formed from the 5,6-dihydrouridin-5-yl
radical (1) under anaerobic conditions is independent of flank-
ing sequence, within experimental error. The 3′-terminal phos-
phate containing product accounts for ∼85% from 5′-32P-16
and 5′-32P-17. The ratios of 24:26a,b in single stranded RNA
resulting from cleavage at the 5′-adjacent nucleotide following
generation of 1 flanked by A (5′-32P-19) or U (5′-32P-18) are
also within experimental error of one another. However, the
absolute yield of 26a,b from the single stranded substrates is
<1%, and the variation is large. Although this is discussed in
more detail below, these observations suggest that the 3′-
phosphate (24) and 3′-deoxy-2′-keto (26a,b) products are derived
from a common intermediate that differs only with respect to the
nucleobase present at the strand scission site (A or U), and this
remote difference does not affect its reactivity.
Effect of pH on Strand Scission and Product
Distribution. The overall strand scission yield resulting from
the 5,6-dihydrouridin-6-yl radical (2) decreased slightly when
the pH was reduced from 7.2 to 3.8. However, the ratio of 5′-
fragments containing 3′-deoxy-2′-keto (26a,b) versus 3′-
phosphate (24) termini increased significantly. The effect was pro-
posed to result from an influence of the acidity on the reactivity
of a cation radical intermediate (5, Scheme 1) formed upon
strand scission via β-elimination of the C2′-radical (4) resulting
from hydrogen atom abstraction by the nucleobase radical.
Consequently, the reactivity of the 5,6-dihydrouridin-5-yl
radical (1) in duplex RNA at pH 3.8 was compared to that
at pH 7.2. When the radical was flanked by either A (5′-32P-17)
conditions produced a strand scission product containing a car-
bonyl group at its 3′-terminus. The similarities to the reactivity
of the 5,6-dihydrouridin-6-yl radical (2) suggested that this pro-
duct resulting from reaction at the 5′-adjacent nucleotide was
the 3′-deoxy-2′-keto product (26a,b). An analogous product
Figure 1. MALDI-TOF MS analysis of reaction products from 5,6-dihydrouridin-5-yl (1) in duplex RNA (17) at pH 3.8.
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or U (5′-32P-16) a modest decrease in total strand scission yield
was observed when 1 was generated at pH 3.8 (5′-32P-16, 27.7
contribution to strand scission from hydrogen atom abstraction
from another ribose position on the 5′-adjacent nucleotide.
Hydrogen atom abstraction from the C1′-position would not
yield a direct strand break, and examination of molecular mod-
els suggested that the C4′- and C5′-hydrogen atoms were too
far. The C3′-hydrogen atom of the 5′-adjacent nucleotide is in
close proximity to 1 in duplex RNA, and the C3′-H bond dis-
sociation energy has been calculated with the ONIOM-G3B3
approach to be ∼93 kcal/mol, which is comparable to the bond
strength of the C1′-carbon−hydrogen bond, albeit considerably
higher than the C2′-carbon−hydrogen bond.34 3′-Deuterated
uridine was incorporated via its respective phosphoramidite at
the 5′-adjacent nucleotide (21), which was synthesized from
known ketone 29.33,35 However, it had no effect on strand
scission resulting from 1 at either pH 7.2 or 3.8 (Table 3),
indicating that the smaller observed KIE on strand scission
emanating from the 5,6-dihydrouridin-5-yl radical (1) than
from 2 is not due to abstraction of other hydrogen atoms from
the 5′-adjacent nucleotide.
2.7%; 5′-32P-17, 21.4
1.3%) compared to 7.2 (Table 2).
However, in both sequences there was a large increase in
the relative amount of 3′-deoxy-2′-keto product (26a,b) com-
pared to the internucleotidyl cleavage product containing a
3′-phosphate (24, Figure 2). The changes in product ratios
Figure 2. Effect of pH on 3′-termini of strand scission products from
5,6-dihydrouridin-5-yl (1) in duplex RNA.
resulted from an increase in the yield of 26 and a decrease in
the amount of 24 formed at pH 3.8. As discussed further below,
this is consistent with the partitioning of the cation radical in-
termediate (5, Scheme 1) formed via β-elimination of the C2′-
radical (4, Scheme 1) at the 5′-adjacent site.
A Common Mechanism for Strand Scission from 5,6-
Dihydrouridin-5-yl (1) and 5,6-Dihydrouridin-6-yl (2)
Radicals. Although an explanation for the smaller effect of C2′-
deuteration on strand scission from 5,6-dihydrouridin-5-yl
radical (1) than from 2 is discussed below, the deuterium iso-
tope effect observed is consistent with the involvement of
hydrogen atom abstraction from this position in strand scission.
In addition, the products formed and pH effects on strand scis-
sion under anaerobic conditions also suggest that 5′-inter-
nucleotidyl strand scission from 1 and the 5,6-dihydrouridin-6-yl
radical (2) occur via a common intermediate, the C2′-radical
(4, Schemes 1, 3). The C2′-radical results from hydrogen atom
abstraction by the 5,6-dihydrouridin-5-yl radical (1) and under-
goes β-elimination to produce a strand break and the cation
radical (5) concomitantly. Olefin cation radical formation from
α-heteroatom stabilized radicals containing leaving groups in
the β-position is precedented in nucleic acid chemistry and
small molecules.25,36−39 Based upon the final products and the
pH effect on their formation, we previously proposed com-
peting deprotonation pathways leading from 5.10 The data
obtained involving the 5,6-dihydrouridin-5-yl radical (1) are
consistent with this as well (Scheme 3b,c).
Effect of Thiol (BME) on Strand Scission and Product
Distribution. An alternate possibility for the smaller KIE
observed from 1 compared to the 5,6-dihydrouridin-6-yl radical
(2) is that the former is more reactive and gives rise to an
earlier transition state. The ability of the 5,6-dihydrouridin-5-yl
radical (1) to abstract hydrogen atoms from the ribose back-
bone was evaluated by measuring the strand break yield
produced from the anaerobic photolysis of 5′-32P-16 over a
range of BME concentrations (eq 3).
Effects of Deuterium Substitution on Strand Scission
in Duplex RNA Resulting from the 5,6-Dihydrouridin-5-yl
Radical (1). Proximity effects, bond dissociation energies, and
similarities to products formed by the 5,6-dihydrouridin-6-yl
radical (2) discussed above suggested that 1 abstracts the C2′-
hydrogen atom en route to strand scission at the 5′-adjacent
nucleotide. Indeed, C2′-deuteration of the 5′-adjacent uridine
(20) resulted in a significant reduction in strand scis-
sion at that site. At pH 7.2 the total strand scission at the 5′-
adjacent nucleotide was reduced from slightly less than 32% to
less than 9%, giving rise to an apparent KIE = 3.6. Although
not rigorously a kinetic isotope effect, the deuterium KIEs
reported here (Table 3) are defined by the ratio of cleavage at
Table 3. Deuterium Isotope Effect on the Reactivity of 5,6-
Dihydrouridin-5-yl (1) in Duplex RNA
a
KIE
pH
C2′-2H
C3′-2H
7.2
3.8
3.6 0.7 (5)
4.7 0.8 (3)
1.1 0.1 (3)
0.9 0.1 (2)
a
KIE determined by measuring cleavage in 5′-32P-16 relative to that in
5′-32P-20 or 5′-32P-21. Each experiment was carried out in triplicate,
and the number of experiments is in parentheses.
the 5′-adjacent nucleotide to 1 in 5′-32P-16 to that in 5′-32P-20
(or 5′-32P-21) exposed to the same photolysis conditions. The
average KIE at pH 3.8 is larger but statistically within error of
the effect at higher pH. Although the KIE at both pH’s were
significant, the effect of C2′-deuteration on strand scission from
the 5,6-dihydrouridin-5-yl radical (1) compared to 2 was con-
siderably smaller.9
k
[1][BME]
[Trapped Prod. ]
[Cleaved Prod. ]
Trap
The smaller C2′-2H KIE observed for the 5,6-dihydrouridin-
5-yl radical (1) compared to 2 may have arisen due to a
=
k
[1]
(3)
Cleave
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Scheme 3
Figure 3. Effect of BME on the 3′-deoxy-2′-keto product (26a) from
5,6-dihydrouridin-5-yl (1) in duplex RNA.
Computational Analysis of the Reactivity of the 5,6-
Dihydrouridin-5-yl (2) and 5,6-Dihydrouridin-6-yl (1)
Radicals. The observations that 1 exhibits a smaller KIE than 2
and the need for higher thiol concentrations to quench strand
scission from the former suggest that 5,6-dihydrouridin-5-yl (1)
is the more reactive of the two radicals. Previous calculations
for dihydrothymine at the Hartree−Fock (ROHF) level using
the isodesmic reaction approach predicted that the C6-nucleo-
base radical was more stable than the regioisomeric C5-radical
with the C5−H bond (93 kcal/mol) about 4 kcal/mol larger
than the C6−H bond (89 kcal/mol).42 We have calculated the
bond dissociation enthalpies of the C5- and C6-carbon−hydro-
gen bonds of the more pertinent N1-methyl-5,6-dihydrouracil
model system with the thermodynamically predictive G3B3
approach using Gaussian09.43 The G3B3 calculated C5−H
bond dissociation enthalpy (95.3 kcal/mol) was 2.8 kcal/mol
larger than the respective C6-bond (92.5 kcal/mol). If all of the
predicted 2.8 kcal/mol difference in bond dissociation enthalpy
were translated to the respective transition states for hydrogen
atom abstraction from the C2′-position of the 5′-adjacent nucle-
otide, the 5,6-dihydrouridin-5-yl radical (2) would react more
than 125 times faster than the C6-radical. A less than 2 kcal/
mol difference in activation energies would account for the
minimum (24-fold) difference in kCleave for 1 and 2.
Which Step Is Rate Determining in Cleavage from the
5,6-Dihydrouridin-5-yl Radical (1)? The greater rate con-
stant for strand scission via the 5,6-dihydrouridin-5-yl radical
(1) compared to 2 leaves the rate determining step uncertain
(H•abs or β-Elim in Scheme 3). There is only one study in
which the rate constant for a comparable reaction was exam-
ined in a nucleic acid. Giese estimated that the C4′-radical in
duplex DNA eliminates the 3′-phosphate with a rate constant of
∼100 s−1 and approximately an order of magnitude faster in
single stranded material.44 DNA lesions also cleave faster in
single stranded material.45,46 In contrast, cleavage from the 5,6-
dihydrouridin-6-yl radical (2) (kCleave) was less than 100 s−1 in
either single or double stranded RNA and strand scission was
faster in the latter substrate.10 The dependency of kCleave on sec-
ondary structure indicated that C2′-radical (4) cleavage could
not be the rate limiting step. The rate constant for strand scis-
sion from the 5,6-dihydrouridin-5-yl radical (1) is considerably
faster, and from these data alone it is not possible to determine
whether the formation of 4 or subsequent elimination from it is
the rate determining step. Independent generation of the C2′-
radical (4) would be very useful for determining the rate con-
stant for β-elimination to form 5.
Subtracting the percentage of cleaved RNA in the presence
of a given BME concentration from that in the absence of thiol
provided the yield of radical trapped by the hydrogen atom
donor. Similarly, the amount of strand scission at high BME
concentration (5 mM) was subtracted from the corresponding
amount of that product formed in the presence of a lower thiol
concentration (or the absence of thiol) in order to account for
any strand scission not due to 1. The ratio of trapped product/
cleaved product varied linearly with BME concentration, and
the slope of this line (3.5 × 103 M−1) corresponded to kTrap
/
kCleave. A similar study on the reactivity of the 5,6-dihydro-
uridin-6-yl radical (2) (kTrap/kCleave = 8.4 × 104 M−1) produced
a rate constant for strand scission, kCleave = 31
10 s−1, by
assuming that kTrap = (2.6 0.5) × 106 M−1s−1, which is the
approximate rate constant for BME reaction with monomeric 2
that was determined using competitive kinetics.10,23 Due to the
slow rate constant for strand scission from 2, 0.1 mM BME was
able to prevent this process. In contrast, millimolar BME con-
centrations were required to prevent strand scission from the
5,6-dihydrouridin-5-yl radical (1). The rate constant for BME
trapping of 1 is unknown, and that for 2 is in the lower range
for reactions of thiols with alkyl radicals, which are typically
∼8 × 106 M−1 s−1.40 If we assume that the 5,6-dihydrouridin-6-yl
radical (2) and 1 have equal rate constants for their reactions
with BME, then the latter’s rate constant for cleavage in double
stranded RNA (kCleave) is 24 times greater (744 s−1). This rate
constant is a minimum and could be as high as 2.3 × 103 s−1 if
the 5,6-dihydrouridin-5-yl radical (1) reacts with BME at rates
that are more typical for an alkyl radical.
Although the overall amount of strand scission is inversely
proportional to BME concentration, the effect of the thiol on
the distribution of end group products is more complicated.
The absolute yield of the 3′-deoxy-2′-keto product (26a) in-
creases in the presence of low BME concentrations at the
expense of a 3′-phosphate containing fragmentation product,
reaching a maximum at ∼25−500 μM (Figure 3). BME has a
similar effect on 26a formed from the 5,6-dihydrouridin-6-yl
radical (2).33 We propose that the thiol reduces the olefin cation
radical (5) produced upon strand scission (Scheme 3a).41
3922
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Article
CONCLUSIONS
ACKNOWLEDGMENTS
■
■
The 5,6-dihydrouridin-5-yl radical (1) is the second nucleobase
radical shown by independent generation from chemical pre-
cursors to lead to direct strand scission in RNA. Nucleobase
radicals are the major products of the reactions between OH•
and pyrimidines in RNA, and radiation chemists have used a
variety of tools to study their reactivity.5 The preference for
producing direct strand breaks in double stranded compared to
single stranded RNA by 1 is even greater than that of the 5,6-
dihydrouridin-6-yl radical (2), and it is more reactive. It is
interesting to note that scientists correctly inferred that C5-
radicals would be more reactive than the C6-radicals that are
produced in greater amounts from experiments in which the re-
active species were generated randomly.14 Computational
experiments support the greater reactivity of the 5,6-dihydro-
uridin-5-yl radical (1) vs 2, and the approximate rate constants
for cleavage (kCleave) and KIEs are also in agreement with this.
The 5,6-dihydrouridin-5-yl radical (1) exhibits a higher rate
constant for strand scission and a smaller KIE than 2, which are
consistent with an earlier transition state, as would be expected
based upon the computational experiments.
Strand scission via nucleobase radicals in RNA is driven
by the relatively weak C2′-carbon−hydrogen bond dissociation
energy, which provides a viable pathway for transferring spin to
the carbohydrate backbone.34 This pathway is absent in DNA,
and it is reflected in the inability of DNA nucleobase radicals to
produce direct strand breaks efficiently.19,20,28,47,48 The
reactivity of RNA nucleobase radicals is contrary with respect
to previous observations that indicated that it was more stable
to oxidative stress than DNA.49 However, these studies focused
on direct oxidation of the carbohydrate backbone. For instance,
C4′-radicals lead to strand scission more efficiently in DNA
than in RNA.50 Our studies on pyrimidine radicals suggest that
RNA is more susceptible to strand scission when nucleobase
radicals are formed.
M.M.G. and M.J.E.R. are grateful for support of this research by
the National Institute of General Medical Sciences (GM-054996).
M.J.E.R. thanks the NIGMS for a Research Supplement to Pro-
mote Diversity in Health-Related Research. M.D.S. and V.B.
thank the National Cancer Institute for support (CA-045424).
MALDI-TOF MS data were collected on an instrument pur-
chased with support from the NSF-0840463.
REFERENCES
■
(1) Adilakshmi, T.; Bellur, D. L.; Woodson, S. A. Nature 2008, 455,
1268−1272.
(2) Ralston, C. Y.; Sclavi, B.; Sullivan, M.; Deras, M. L.; Woodson, S.
A.; Chance, M. R.; Brenowitz, M. Methods Enzymol. 2000, 317,
353−368.
(3) Tullius, T. D.; Greenbaum, J. A. Curr. Opin. Chem. Biol. 2005, 9,
127−134.
(4) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
& Francis: London, 1987.
(5) von Sonntag, C. Free-Radical-Induced DNA Damage and Its
Repair; Springer-Verlag: Berlin, 2006.
(6) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9738−9743.
(7) Dussy, A.; Meggers, E.; Giese, B. J. Am. Chem. Soc. 1998, 120,
7399−7403.
́
(8) Pitie, M.; Pratviel, G. Chem. Rev. 2010, 110, 1018−1059.
(9) Jacobs, A. C.; Resendiz, M. J. E.; Greenberg, M. M. J. Am. Chem.
Soc. 2010, 132, 3668−3669.
(10) Jacobs, A. C.; Resendiz, M. J. E.; Greenberg, M. M. J. Am. Chem.
Soc. 2011, 133, 5152−5159.
(11) Melvin, T.; Butchway, S. W.; Parker, A. W.; O’Neill, P. J. Am.
Chem. Soc. 1996, 118, 10031−10036.
(12) Deeble, D. J.; Schulz, D.; Von Sonntag, C. Int. J. Radiat. Biol.
1986, 49, 915−926.
(13) Lemaire, D. G. E.; Bothe, E.; Schulte-Frohlinde, D. Int. J. Radiat.
Biol. 1987, 51, 319−330.
(14) Hildenbrand, K.; Schulte-Frohlinde, D. Int. J. Radiat. Biol. 1989,
55, 725−738.
Upon completing studies on the reactivity of the 5,6-dihydro-
uridin-6-yl radical (2) we proposed that the preference for
strand scission in double stranded versus single stranded RNA
under anaerobic conditions may be useful for distinguishing
between single and double stranded RNA by comparing cleav-
age in hydroxyl radical reactions under aerobic and anaerobic
conditions.10 We also suggested that enhanced cleavage under
anaerobic conditions by 2 was contrary to the generally accept-
ed oxygen enhancement by ionizing radiation and that nucleo-
base radicals are not major contributors to direct strand scission
under aerobic conditions.4 The results described herein rein-
force these notions.
(15) Jones, G. D. D.; O’Neill, P. Int. J. Radiat. Biol. 1991, 59, 1127−
1145.
(16) Schulte-Frohlinde, D.; Opitz, J.; Gorner, H.; Bothe, E. Int. J.
̈
Radiat. Biol. 1985, 48, 397−408.
(17) Catterall, H.; Davies, M. J.; Gilbert, B. C. J. Chem. Soc., Perkin
Trans. 2 1992, 1379−1385.
(18) Giese, B. Annu. Rev. Biochem. 2002, 71, 51−70.
(19) Greenberg, M. M. In Wiley Series of Reactive Intermediates in
Chemistry and Biology; Greenberg, M. M., Ed.; John Wiley and Sons:
2009; Vol. 2, pp 135−162.
(20) Greenberg, M. M. Org. Biomol. Chem. 2007, 5, 18−30.
(21) Bales, B. C.; Kodama, T.; Weledji, Y. N.; Pitie, M.; Meunier, B.;
Greenberg, M. M. Nucleic Acids Res. 2005, 33, 5371−5379.
(22) Kroeger, K. M.; Hashimoto, M.; Kow, Y. W.; Greenberg, M. M.
Biochemistry 2003, 42, 2449−2455.
ASSOCIATED CONTENT
■
(23) Newman, C. A.; Resendiz, M. J. E.; Sczepanski, J. T.; Greenberg,
M. M. J. Org. Chem. 2009, 74, 7007−7012.
S
* Supporting Information
Experimental procedures, thiol effects on products, mass
spectral data, and sample autoradiograms. Complete ref 43.
These materials are available free of charge via the Internet at
http://pubs.acs.org.
(24) Koerner, S.; Bryant-Friedrich, A.; Giese, B. J. Org. Chem. 1999,
64, 1559−1564.
(25) Giese, B.; Beyrich-Graf, X.; Erdmann, P.; Petretta, M.; Schwitter,
U. Chem. Biol. 1995, 2, 367−375.
(26) Tronche, C.; Goodman, B. K.; Greenberg, M. M. Chem. Biol.
1998, 5, 263−271.
AUTHOR INFORMATION
■
(27) Barvian, M. R.; Greenberg, M. M. J. Org. Chem. 1995, 60, 1916−
1917.
Corresponding Author
mgreenberg@jhu.edu
(28) Carter, K. N.; Greenberg, M. M. J. Am. Chem. Soc. 2003, 125,
13376−13378.
Notes
(29) Carter, K. N.; Greenberg, M. M. J. Org. Chem. 2003, 68, 4275−
4280.
The authors declare no competing financial interest.
3923
dx.doi.org/10.1021/ja300044e | J. Am. Chem. Soc. 2012, 134, 3917−3924

Journal of the American Chemical Society
Article
(30) Lahoud, G.; Fancher, J.; Grosu, S.; Cavanaugh, B.; Bryant-
Friedrich, A. Bioorg. Med. Chem. 2006, 14, 2581−2588.
(31) Hayakawa, H.; Tanaka, H.; Miyasaka, T. Tetrahedron 1985, 41,
1675−1683.
(32) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N.
Nucleic Acids Res. 1995, 23, 2677−84.
(33) See Supporting Information.
(34) Li, M.-J.; Liu, L.; Wei, K.; Fu, Y.; Guo, Q.-X. J. Phys. Chem. B
2006, 110, 13582−13589.
(35) Serafinowski, P. J.; Barnes, C. L. Tetrahedron 1996, 52, 7929−
7938.
(36) Giese, B.; Beyrich-Graf, X.; Erdmann, P.; Giraud, L.;
Imwindelried, P.; Muller, S. N.; Schwitter, U. J. Am. Chem. Soc.
̈
1995, 117, 6146−6147.
(37) Dizdaroglu, M.; Von Sonntag, C.; Schulte-Frohlinde, D. J. Am.
Chem. Soc. 1975, 97, 2277−2278.
(38) Newcomb, M.; Miranda, N.; Huang, X.; Crich, D. J. Am. Chem.
Soc. 2000, 122, 6128−6129.
(39) Horner, J. H.; Taxil, E.; Newcomb, M. J. Am. Chem. Soc. 2002,
124, 5402−5410.
(40) Newcomb, M. Tetrahedron 1993, 49, 1151−1176.
(41) Burke, M.; Edge, R.; Land, E. J.; McGarvey, D. J.; Truscott,
T. G. FEBS Lett. 2001, 500, 132−136.
(42) Colson, A.-O.; Becker, D.; Eliezer, I.; Sevilla, M. D. J. Phys.
Chem. A 1997, 101, 8935−8941.
(43) Frisch, M. J.; et al. Gaussian 09; Gaussian, Inc.: Wallingford CT,
2009.
(44) Giese, B.; Dussy, A.; Meggers, E.; Petretta, M.; Schwitter, U.
J. Am. Chem. Soc. 1997, 119, 11130−11131.
(45) Kim, J.; Gil, J. M.; Greenberg, M. M. Angew. Chem., Int. Ed.
2003, 42, 5882−5885.
(46) Chen, J.; Stubbe, J. Biochemistry 2004, 43, 5278−5286.
(47) Hong, I. S.; Carter, K. N.; Greenberg, M. M. J. Org. Chem. 2004,
69, 6974−6978.
(48) Hong, I. S.; Carter, K. N.; Sato, K.; Greenberg, M. M. J. Am.
Chem. Soc. 2007, 129, 4089−4098.
(49) Thorp, H. H. Chem. Biol. 2000, 7, R33−R36.
(50) Crich, D.; Mo, X.-S. J. Am. Chem. Soc. 1997, 119, 249−250.
3924
dx.doi.org/10.1021/ja300044e | J. Am. Chem. Soc. 2012, 134, 3917−3924