Oxygen independent DNA interstrand cross-link formation by a nucleotide radical

Article abstract of DOI:10.1021/ja0563657

A 5-(2′-Deoxyuridinyl)methyl radical (1) was independently generated from three photochemical precursors and is the first example of a DNA radical that forms interstrand cross-links. Oxygen labeling experiments support generation of 1 by all precursors. I

Full text of DOI:10.1021/ja0563657

Published on Web 12/20/2005
Oxygen Independent DNA Interstrand Cross-Link Formation
by a Nucleotide Radical
In Seok Hong, Hui Ding, and Marc M. Greenberg*
Contribution from the Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles
Street, Baltimore, Maryland 21218
Received September 15, 2005; E-mail: mgreenberg@jhu.edu
Abstract: A 5-(2′-Deoxyuridinyl)methyl radical (1) was independently generated from three photochemical
precursors and is the first example of a DNA radical that forms interstrand cross-links. Oxygen labeling
experiments support generation of 1 by all precursors. Interstrand cross-links are produced upon irradiation
of DNA containing any of the precursors. Cross-linking occurs via reaction with the opposing 2′-
deoxyadenosine and is independent of O2. The independence of cross-link formation on O2 is explained
by kinetic analysis, which shows that the radical reacts reversibly with O2. Examination of the effects of
glutathione on cross-link formation under anaerobic conditions suggests that adoption of the syn-
conformation by 1 is the rate-limiting step in the process. Interstrand cross-link formation is reversible in
the presence of a good nucleophile. The stability of the interstrand cross-link suggests that the isolated
molecule is a rearrangement product of that formed in solution. The rearrangement is a consequence of
the isolation procedure but also occurs slowly in solution. Oxygen independent cross-link formation may
be useful for the purposeful damage of DNA in hypoxic tumor cells, where O2 is deficient.
The biological importance of DNA damage is evident in many
ways. The double sword nature of these chemical transforma-
tions is illustrated by their involvement in the etiology and
treatment of cancer.1-3 In addition, there is a growing apprecia-
tion of the importance of DNA damage in aging.4-6 DNA
radicals are an important family of reactive intermediates that
give rise to modified intact polymers and also lead to single-
and double-strand breaks.7-9 Recently, we reported that an
independently generated 5-(2′-deoxyuridinyl)methyl radical (1)
produced interstrand cross-links (ISCs) with an opposing 2′-
deoxyadenosine (dA) (Scheme 1).10 The radical is derived from
formal hydrogen atom abstraction from the methyl group of
thymidine and is not believed to be formed in large amounts
by ionizing radiation despite the low bond dissociation energy
(BDE) of the carbon-hydrogen bonds and their accessibility
in the major groove. The preliminary report concerning 1 was
the first explicit example of DNA interstrand cross-link (ISC)
formation through a nucleic acid radical. ISCs are often
responsible for the cytotoxic effects of DNA damaging agents.11,12
Hence, ISC formation by a radical produced in even small
amounts (e.g., 1) by agents such as γ-radiolysis could be
biologically significant. Our interest in the radical-mediated
cross-link formation is enhanced by the observation that the
process involving 1 is independent of O2. This suggests that, in
addition to being chemically novel, the formation of interstrand
cross-links from 1 may be useful in hypoxic cells, such as those
present in tumors.13 DNA damaging agents, such as γ-radiolysis,
are less efficient in hypoxic cells because many of the chemical
pathways require O2. Herein we describe experiments that
further elucidate and substantiate this novel mechanism for DNA
interstrand cross-link formation.
In addition to arising from direct hydrogen atom abstraction,
the 5-(2′-deoxyuridinyl)methyl radical (1) can be formed from
a two-step process, such as that induced by the direct effect of
γ-radiolysis (Scheme 1). In accordance with these mechanistic
possibilities, the radical has been implicated as an intermediate
in γ-radiolysis through product studies and in experiments in
which photoinduced electron transfer is used to oxidize thymi-
dine.7,14,15 Oxygenated products for which 1 can serve as a
precursor, most notably 5-formyl-2′-deoxyuridine (9) and 5-hy-
droxymethyl-2′-deoxyuridine (8), have been detected in studies
on the reactivity of thymidine and DNA.16 Prior to the report
by Hong, 1 was independently generated from other photolabile
precursors in a nucleoside, dinucleotide, and single-stranded
oligonucleotides.17,18 Generation of 1 in dinucleotides and single-
(1) DePinho, R. A. Nature 2000, 408, 248-254.
(2) Schar, P. Cell 2001, 104, 329-332.
(3) Loeb, L. A.; Loeb, K. R.; Anderson, J. P. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 776-781.
(4) Kujoth, G. C. et al. Science 2005, 309, 481-484.
(5) Lombard, D. B.; Chua, K. F.; Mostoslavsky, R.; Franco, S.; Gostissa, M.;
Alt, F. W. Cell 2005, 120, 435-567.
(6) Schriner, S. E.; Linford, N. J.; Martin, G. M.; Treuting, P.; Ogburn, C. E.;
Emond, M.; Coskun, P. E.; Ladiges, W.; Wolf, N.; Van Remmen, H.;
Wallace, D. C.; Rabinovitch, P. S. Science 2005, 308, 1909-1911.
(7) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor &
Francis, London, 1987.
(13) Brown, J. M.; Wilson, W. R. Nat. ReV. Cancer 2004, 4, 437-447.
(14) Douki, T.; Cadet, J. Int. J. Radiat. Biol. 1999, 75, 571-581.
(15) Wagner, J. R.; van Lier, J. E.; Johnston, L. J. Photochem. Photobiol. 1990,
52, 333-343.
(16) Dizdaroglu, M.; Jaruga, P.; Birincioglu, M.; Rodriguez, H. Free Radical
Biol. Med. 2002, 32, 1102-1115.
(8) Pogozelski, W. K.; Tullius, T. D. Chem. ReV. 1998, 98, 1089-1107.
(9) Burrows, C. J.; Muller, J. G. Chem. ReV. 1998, 98, 1109-1151.
(10) Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 3692-3693.
(11) Scha¨rer, O. D. Chem. Biol. Chem. 2005, 6, 27-32.
(12) Dronkert, M. L.; Kanaar, R. Mutat. Res. 2001, 486, 217-247.
9
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J. AM. CHEM. SOC. 2006, 128, 485-491
485

A R T I C L E S
Hong et al.
Scheme 1. Generation of, and Interstrand Cross-Link Formation by the 5-(2′-Deoxyuridinyl)methyl Radical (1)
Scheme 2. Trapping Products Produced from the
5-(2′-Deoxyuridinyl)methyl Radical (1)
stranded oligonucleotides led to the formation of tandem
lesions.18 Tandem lesions are defined as two contiguously
damaged nucleotides. Cadet characterized a tandem lesion
derived from 1, involving formation of a covalent between the
radical and C8 of the adjacent 2′-deoxyguanosine. Structurally
similar tandem lesions were also detected when the analogous
radical (5) derived from 5-methyl-2′-deoxycytidine was pro-
duced in a dinucleotide.19 However, in contrast to other
Table 1. Source of Oxygen in 5-(Hydroxymethyl)-2′-deoxyuridine
(8) Determined by Mass Spectrometry
nucleobase radicals, alkali-labile tandem lesions were not
detected by gel electrophoresis when 1 was produced from 2
in duplex DNA.20-22 Interstrand cross-links were the sole
products detected when 1 was produced from 2 in duplex DNA
under aerobic or anaerobic conditions.10 Given this surprising
result, we investigated the mechanism for ISC formation further.
Herein we provide further support that the 5-(2′-deoxyuridinyl)-
methyl radical (1) is responsible for cross-linking. An explana-
tion for why ISC formation is independent of O2 and other issues
are also addressed.
M+
+
2:M+
substrate
H2O/O2
H218O/O2
H2O/18O2
2
11
12
0.05
0.01
0.01
0.08
0.03
0.06
6.1
sigmatropic rearrangement of the selenoxide.24 Formal solvolysis
of 2 would produce a carbocation capable of alkylating dA.
Although the formation of oxygenated thymidine derivatives
was indicative of the intermediacy of 1, isotopic labeling was
used to further develop this connection (Table 1). The source
of the hydroxyl group oxygen atom in 5-hydroxymethyl-2′-
deoxyuridine (8) was determined using 18O-labeled H2O and
O2 in the presence of glutathione (50 mM). The ratio of the
M+ + 2:M+ ions was insignificantly higher when H218O was
used as solvent. However, bubbling 18O2 through the solution
prior to and during photolysis resulted in an approximate 100-
fold increase in the ratio of the M+ + 2:M+ ions, confirming
that O2 is the source of the hydroxyl group oxygen in 8.
Although the O2 trapping data from photolysis of 2 are
consistent with the intermediacy of 1, we sought to strengthen
the argument by developing other precursors to the 5-(2′-
deoxyuridinyl)methyl radical (1). Irradiation of the respective
phenyl sulfides at 254 nm are reported to produce the 5-(2′-
deoxyuridinyl)methyl radical (1) and 5-(2′-deoxycytidinyl)-
methyl radical (5).18,19 Using these molecules as a guide, the
methoxy-substituted derivatives of thymidine (11, 12) were
Results and Discussion
Independent Generation of 5-(2′-Deoxyuridinyl)methyl
Radical (1) from Three Photolabile Precursors. Preliminary
product studies were consistent with independent generation of
1 from the phenyl selenide (2, Scheme 2).23 More complete
studies were undertaken because the radical was not directly
observed and interstrand cross-link formation could be rational-
ized using other electrophilic species as reactants. For example,
1
we recently showed that oxidation of 2 by O2 or NaIO4 also
produces an ISC with the opposing dA following [2,3]-
(17) Anderson, A. S.; Hwang, J. T.; Greenberg, M. M. J. Org. Chem. 2000, 65,
4648-4654.
(18) Romieu, A.; Bellon, S.; Gasparutto, D.; Cadet, J. Org. Lett. 2000, 2, 1085-
1088.
(19) Zhang, Q.; Wang, Y. J. Am. Chem. Soc. 2003, 125, 12795-12802.
(20) Tallman, K. A.; Greenberg, M. M. J. Am. Chem. Soc. 2001, 123, 5181-
5187.
(21) Carter, K. N.; Greenberg, M. M. J. Am. Chem. Soc. 2003, 125, 13376-
13378.
(22) Hong, I. S.; Carter, K. N.; Greenberg, M. M. J. Org. Chem. 2004, 69,
6974-6978.
(23) Hong, I. S.; Greenberg, M. M. Org. Lett. 2004, 6, 5011-5013.
(24) Hong, I. S.; Greenberg, M. M. J. Am. Chem Soc. 2005, 127, 10510-10511.
9
486 J. AM. CHEM. SOC. VOL. 128, NO. 2, 2006

DNA-Radical-Mediated Interstrand Cross-Link Formation
A R T I C L E S
Scheme 3. Reversible Trapping of the 5-(2′-Deoxyuridinyl)methyl
Radical (1) by O2
Figure 1. Product dependence of monomeric 5-(2′-deoxyuridinyl)methyl
radical (1) derived from 12 under aerobic conditions as a function of GSH
concentration.
synthesized with the intent of red-shifting the absorption so that
1 could be generated from them upon irradiation with lamps
that emit maximally at 350 nm. The mono- and dimethoxy-
substituted molecules had λmax (ꢀ, M-1 cm-1) at 258 (6800)
and 267 (5640) nm, respectively. However, the absorbance of
solutions (<100 µM) of each tailed into the g320 nm region,
and the photochemical conversion efficiency of 11 and 12
correlated with their relative absorption. Approximately 50%
of 11 or 12 (50 µM) was converted upon irradiation in a Rayonet
photoreactor for 5 or 30 min, respectively. Product trapping
studies in the presence of GSH and O2 produced the mixture of
oxygenated products and thymidine expected from the formation
of 1. The mass balances (65-76%) were comparable to those
measured for 350 nm photolysis of 2.23 Additional support for
the intermediacy of 1 from the aryl sulfides was acquired by
carrying out the photolyses in H218O and GSH (50 mM),
whereupon no incorporation of 18O in 8 was detected (Table
1).
Table 2. Interstrand Cross-Link Formation as a Function of
Sequence and Radical Precursor
% cross-link
local sequence
(substrate)
aerobic
anaerobic
5′-C2C (14)
5′-G2G (15)
5′-T2T (16)
5′-C11C (17)
5′-C12C (18)
25.7 ( 0.7
12.5 ( 0.5
20.0 ( 0.8
20.7 ( 0.8
20.6 ( 0.2
28.3 ( 0.6
12.6 ( 0.6
20.1 ( 0.7
19.8 ( 0.4
20.6 ( 0.6
used to solve for k-O (3.4 s-1), kGSH (6.9 × 106 M-1 s-1), and
2
kOx (0.08 s-1). This analysis assumed a rate constant for the
trapping of 1 by O2 (2 × 109 M-1 s-1) and that 13 is trapped
by GSH with a rate constant commensurate with that reported
for DNA peroxyl radicals (2 × 102 M-1 s-1).25 The magnitude
of k-O is slightly greater than the respective rate constant
2
Reversible Trapping of 5-(2′-Deoxyuridinyl)methyl Radi-
cal (1) by O2. Product and isotope labeling studies support the
formation of 1 and its trapping by O2 under the 350 nm
photolysis conditions used to generate the radical in DNA.
Oxygenated product formation from monomeric 1 was recon-
ciled with the observation that interstrand cross-linking is
independent of dioxygen by examining the possibility that O2
trapping is reversible. We probed for reversible formation of
monomeric 13 (Scheme 3) by measuring the distribution of
radical trapping products as a function of GSH concentration.
The product dependence on GSH concentration was established
by assuming that thymidine (6) is formed via reaction between
1 and GSH with bimolecular rate constant kGSH. Oxygenated
products were assumed to emanate from 13 upon its reaction
with GSH (kOO•) or by other processes (kOx, Scheme 3, eq 1).
determined for the peroxyl radical derived from the C4′-DNA
radical but is within the range expected for such reactions.26,27
The value of the rate constant determined for reaction between
1 and GSH (kGSH) is within the realm of what one would expect
for reaction between a thiol and an alkyl radical.28 This
calibration suggests that the value determined for k-O is a
2
reasonable estimate.
Interstrand Cross-Linking by a Common Intermediate
Produced from Three Photolabile Molecules. The above
experiments demonstrate that the phenyl selenide (2) and aryl
sulfides (11, 12) produce the nucleoside radical upon 350 nm
photolysis. Further evidence was sought to support that the 5-(2′-
deoxyuridinyl)methyl radical (1) was responsible for DNA
interstrand cross-links. Concern that ISC formation was the
result of an excited-state reaction between the phenyl selenide
(2) and a neighboring nucleotide was minimized by showing
that duplexes containing 2 (14-16) produce cross-links (Table
2).29 The ISC yield was significantly lower when the radical
precursor was flanked by dG. However, lower yields were not
accompanied by other products such as direct strand breaks or
alkali-labile lesions. The lower ISC yield was not a result of
If O2 trapping (kO ) is reversible and one assumes that the
2
peroxyl radical (13) is formed under steady-state conditions,
the ratio of thymidine (6) to oxygenated products (7-10, TOx
)
should vary nonlinearly with GSH concentration (Scheme 3,
eq 2). Under conditions where peroxyl radical trapping
(kOO•[GSH]) is much greater than the rate constant describing
loss of O2 from 13 (k-O ), the product ratio is expected to exhibit
2
(25) Hildenbrand, K.; Schulte-Frohlinde, D. Int. J. Radiat. Biol. 1997, 71, 377-
385.
a linear dependence on GSH concentration typical of irreversible
O2 trapping. Indeed, the nonlinear dependence of the ratio of
thymidine to oxygenated products is evident at low thiol
concentrations when the dimethoxy-substituted aryl sulfide (12)
is photolyzed in the presence of GSH between 0.2 mM and 20
mM (Figure 1). Nonlinear regression analysis (R2 ) 0.979) was
(26) Dussy, A.; Meggers, E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 7399-
7403.
(27) von Sonntag, C.; Schuchmann, H.-P. Angew. Chem., Int. Ed. Engl. 1991,
30, 1229-1253.
(28) Newcomb, M. Tetrahedron 1993, 49, 1151-1176.
(29) Adam, W.; Arnold, M. A.; Nau, W. M.; Pischel, U.; Saha-Moeller, C. R.
J. Am. Chem. Soc. 2002, 124, 3893-3904.
9
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A R T I C L E S
Hong et al.
Scheme 4. Interstrand Cross-Link Formation via Methide (20)
Formation
observed upon treatment of 17 or 18 with NaIO4. This provides
further indirect support that photochemical-mediated ISC forma-
tion from 17 and 18 proceeds via the 5-(2′-deoxyuridinyl)methyl
radical (1). Because the phenyl selenide (2) produces cross-
linked DNA upon NaIO4 oxidation, additional measures were
taken to rule this process out. Cross-linking by the methide was
envisioned to involve nucleophilic attack by N1 of dA (Scheme
4) on 20 (although we cannot rule out an SN1 process). An
exogenous nucleophile could compete with dA for 20, but
certainly not with 5-(2′-deoxyuridinyl)methyl radical (1). Reac-
tion of monomeric 2 with NaIO4 (5 mM) in the presence of
NaN3 gave rise to the expected alkyl azide (22), indicating that
azide is compatible with the conditions in which cross-linking
was carried out using this reagent.30 Addition of NaN3 (300
less efficient photochemical conversion of 2. ISC yield does
not increase when photolysis of 15 or 16 is extended to 60 or
90 min, indicating that radical generation is complete within
30 min (data not shown). The lower ISC yield obtained from
the sequence in which 1 is produced when flanked by dGs and
the absence of direct strand breaks or alkali-labile lesions is
consistent with the formation of tandem lesions as described
by Cadet.18 Further support for radical generation from 2 in
DNA is gleaned from ESI-MS analysis following anaerobic
photolysis of 14 in the presence of GSH (50 mM), which
showed that an oligonucleotide containing thymidine is formed
at the expense of ISC.30 Duplexes (17, 18) containing 11 or 12
also produce an ISC upon photolysis in the presence or absence
of O2 (Table 2). Cross-linking generated by the aryl sulfide is
also quenched by GSH.30 Finally, the assumption that cross-
linking occurred exclusively between the radical center and the
opposing dA was verified by examining the hydroxyl radical
cleavage of the cross-link product that was produced from
photolysis of 18.30,31 All of these observations are consistent
with the proposal that photolysis of the respective duplexes
containing 2, 11, and 12 produces interstrand cross-links via a
common intermediate, 5-(2′-deoxyuridinyl)methyl radical (1).
A Methide is Not Responsible for Interstrand Cross-Links
Produced upon Photolysis of Phenyl Selenide (2) or Aryl
Sulfide (11, 12) Containing Duplex DNA. Oxidation of 2 by
mM) to a mixture of 14 and NaIO4 (5 mM) reduced the amount
of cross-linked product by ∼60% regardless of whether the
nucleophile is added before or after the phenyl selenide is
oxidized (Table 3). However, the yield of ISC produced by
photolysis of 14 or 18 was unaffected by this concentration of
NaN3. These data suggest that ISC formation does not proceed
via a methide when DNA containing 2 or 12 is photolyzed and
that 5-(2′-deoxyuridinyl)methyl radical (1) remains the most
viable species responsible for this product.
What Is the Structure of the Cross-Linked Product? The
N6-alkyl adduct (4) was isolated from photolyzed 14 following
enzyme digestion.10 It was postulated that 4 resulted from
rearrangement of an initially formed N1-adduct (3, Scheme 1).
If 3 is the initially formed product, whether it rearranged as a
result of the purification procedure is mechanistically relevant.
The structure of the cross-linked product is also biologically
important because the position in dA that is alkylated could
1
NaIO4 or O2 produces the methide (20), which reacts with
nucleophiles, including the opposing dA in 14 (Scheme 4).24
In contrast, treatment of monomeric 12 with NaIO4 produces a
diastereomeric mixture of sulfoxide 21, which does not rearrange
even upon heating to 90 °C. This observation is consistent with
Table 3. Effect of NaN3 on Interstrand Cross-Linking in Duplexes
Subjected to Photolysis or NaIO4 Conditions
% cross-linking
the expected higher barrier for a [2,3]-sigmatropic rearrangement
faced by an allylic sulfoxide compared to that for a similar
selenoxide.32 As expected, based upon this result, ISC is not
duplex
method
no NaN3
NaN3 (before oxid)
NaN3 (after oxid)
14
14
18
hν
NaIO4
hν
29.9 ( 1.0
49.2 ( 0.2
18.7 ( 1.4
32.1 ( 1.5a
21.7 ( 0.7a
17.5 ( 0.4b
29.3 ( 0.5a
18.6 ( 0.4a
(30) See Supporting Information.
(31) Millard, J. T.; Weidner, M. F.; Kirchner, J. J.; Ribeiro, S.; Hopkins, P. B.
Nucleic Acids Res. 1991, 19, 1885-1892.
a [NaN3] ) 0.3 M. b [NaN3] ) 0.1 M.
9
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DNA-Radical-Mediated Interstrand Cross-Link Formation
A R T I C L E S
Figure 2. Stability of ISC from 14 in the absence and presence of NaN3 (0.3 M) or GSH (5 mM). (A) Photochemically generated ISC. (B) NaIO4 generated
ISC. No additive (9), GSH (2), NaN3 (b).
Scheme 5. Detecting Reversibility of Interstrand Cross-Linking via Trapping with Azide
affect how the lesion is repaired.33,34 We sought to determine
if the ISC product in solution is 3 or 4 by taking advantage of
reversible alkylation of dA at N1.35 N6-Alkylation is irreversible
under the mild incubation conditions used in this study. Duplex
14 was cross-linked under photochemical conditions and by
reaction with NaIO4 in side-by-side reactions.24 After precipita-
tion of the sodium periodate reaction, the cross-linked products
were incubated at 37 °C under identical buffer conditions in
the absence and presence of NaN3 (0.3 M) or GSH (5 mM).
Aliquots were removed over the course of 4.6 days. In the
absence of azide, ISC product(s) were stable regardless of how
they were produced (Figure 2). The ISC product(s) were also
stable in the presence of GSH (5 mM), which is present at a
similar concentration in cells. However, in the presence of NaN3
the amount of ISC products decreased steadily (Figure 2) and
followed first-order kinetics.30 The rate constant for decomposi-
tion of the cross-linked DNA produced via photolysis was (2.9
( 0.1) × 10-6 s-1 (t1/2 ) 66.0 h). The ISC generated by
periodate decomposed at a rate that was within experimental
error of the photochemically generated cross-linked DNA ((3.0
( 0.1) × 10-6 s-1, t1/2 ) 64.2 h). The similarity in the observed
rate constants suggests that the cross-links produced by the two
reaction conditions are the same. Moreover, the reversibility of
the ISCs suggest that 4 is produced during the isolation
procedure. Molecular modeling and the inherent nucleophilicity
of N1 in dA lead us to suggest that 3 is the initial cross-linked
product formed in duplex DNA from syn-5-(2′-deoxyuridinyl)-
methyl radical (1) or the methide (20).10,35
The disappearance of the cross-link product in the presence
of azide can be explained via rate-limiting formation of an ion
pair (IP, Scheme 5) or direct attack by the nucleophile on 3. In
principle, these mechanisms are distinguishable from one
another by measuring the observed rate constant for the
disappearance of ISC as a function of azide concentration. Rate-
limiting formation of an ion pair requires there to be a nonlinear
dependence of kObs (eq 3) on azide concentration, provided that
k-1 + k-2 are neither much greater nor much smaller than
kAz[Az]. The rate constant for direct nucleophilic attack on the
initially formed ISC product would vary linearly with azide
concentration. We found that kObs varied linearly when [Az]
varied from 50 to 500 mM (Figure 3). These data suggest that
either azide reacts directly with 3 in the rate-determining step
or that the relative magnitudes of k-1, k-2, and kAz[Az] prevent
(32) Reich, H. J.; Yelm, K. E.; Wollowitz, S. J. Am. Chem. Soc. 1983, 105,
2503-2504.
(33) David, S. S.; Williams, S. D. Chem. ReV. 1998, 98, 1221-1261.
(34) Stivers, J. T.; Jiang, Y. L. Chem. ReV. 2003, 103, 2729-2759.
(35) Veldhuyzen, W. F.; Shallop, A. J.; Jones, R. A.; Rokita, S. E. J. Am. Chem.
Soc. 2001, 123, 11126-11132.
9
J. AM. CHEM. SOC. VOL. 128, NO. 2, 2006 489

A R T I C L E S
Hong et al.
Figure 5. Ratio of ssDNA:ISC produced from photolysis of 14 as a function
of GSH concentration.
Figure 3. Dependence of the observed rate constant for the disappearance
of ISC (kObs) on NaN3 concentration.
How Fast Is Interstrand Cross-Link Formation by the
5-(2′-Deoxyuridinyl)methyl Radical (1)? The rate constant for
ISC formation (kISC) was estimated by measuring the depen-
dence of its formation on the concentration of GSH under
anaerobic conditions (Scheme 6, eq 4). This method assumes
that the photochemical precursor (2) is completely consumed
and that single-stranded products result from GSH trapping of
1 (kGSH) and other pathways that do not involve thiol (kX). The
lack of an effect of extended irradiation (up to 90 min) on ISC
yield noted above attests to the complete photochemical
conversion of 2. The ratio of single-stranded product (ssDNA)
to ISC obtained from photolysis of 14 varies linearly as a
function of GSH concentration (Figure 5). The estimated rate
constant for ISC formation from the 5-(2′- deoxyuridinyl)methyl
radical (1) is calculated to be 3.8 × 104 s-1 or 2.6 × 104 s-1
(R2 ) 0.995), depending upon whether one uses the upper limit
for alkyl radical trapping by a thiol (1 × 107 M-1 s-1) from the
literature or that estimated from Figure 1 (6.9 × 106 M-1 s-1),
respectively.28
Figure 4. ISC stability to heating (90 °C, 30 min) upon incubation in
aqueous buffer.
detecting the nonlinear dependence of kObs on azide ion
concentration. Carrying out experiments in significantly higher
concentrations of azide was deemed to be unfeasible.
Interstrand cross-link formation requires that 1 adopt the syn-
conformation and cannot be faster than rotation about the
glycosidic bond (Scheme 7). The estimated rate constants for
ISC formation from 1 are faster than what one might expect
based upon NMR determination of A‚T base pair dynamics.
Although A‚T base pair lifetimes less than 1 ms have been
Although cross-linking through N1 of dA (3) is the primary
product, the ISC decomposition studies (Figure 2) do not
eliminate the possibility that 3 is slowly converted to 4.
Qualitative evidence for this was extracted by exploiting the
anticipated difference in stability of the cross-link products.
Although N6-alkylated deoxyadenosines are thermally stable,
N1-alkylation is reversible.35-37 Therefore, we assumed that 3
would at least partially revert to single-stranded material upon
heating. Indeed, heating a freshly cross-linked sample at 90 °C
for 30 min converts approximately 25% of the product to single-
stranded DNA (Figure 4). Similar treatment of aliquots of
photochemically cross-linked material incubated at 37 °C taken
over the course of 4 days revealed that the fraction of material
converted to single-stranded DNA decreased over time (Figure
4), indicating that 3 does isomerize to 4 in solution. These data
suggest that it will be important to elucidate the effects of 3
and 4 on DNA repair and replication.
detected, typical values are in the millisecond (kIsom ∼103 s-1
)
range.38,39 Some factors that might help explain this difference
are that the cross-linking experiments are carried out at higher
temperature and lower pH. In addition, we cannot discount the
possibility that the phenyl selenide precursor produces the 5-(2′-
deoxyuridinyl)methyl radical (1) in a conformation that facili-
tates adoption of the syn-conformation.
Conclusions
A 5-(2′-deoxyuridinyl)methyl radical (1) was generated from
three different photochemical precursors in a variety of sequence
contexts. In all instances the radical produces interstrand cross-
links (ISCs). ISC formation is independent of O2 because
peroxyl radical formation (13) is reversible. Analysis of the
stability of the ISC product indicates that the product resulting
from radical addition to N1 of dA is the primary product but
that this molecule isomerizes in solution to the thermodynami-
cally more stable N6-adduct.35-37
(36) Veldhuyzen, W. F.; Parde, P.; Rokita, S. E. J. Am. Chem. Soc. 2003, 125,
14005-14013.
(37) Zhou, Q.; Rokita, S. E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15452-
15457.
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490 J. AM. CHEM. SOC. VOL. 128, NO. 2, 2006

DNA-Radical-Mediated Interstrand Cross-Link Formation
A R T I C L E S
Scheme 6. Competition between ISC Formation and GSH Trapping of 1
Scheme 7. ISC Formation via Rotation about the Glycosidic Bond in the 5-(2′-Deoxyuridinyl)methyl Radical (1)
The 5-(2′-Deoxyuridinyl)methyl radical (1) is the first
example of a DNA radical that produces interstrand cross-links.
ISC formation in the absence of O2 is potentially important given
the hypoxic nature of tumor cells. The important biological
effects of other cross-links provide the impetus to determine
the consequences of 3, as well as its yield in DNA exposed to
various forms of oxidative stress. In addition, the accessibility
and low BDE of the respective carbon-hydrogen bond in
thymidine make 1 an attractive target for synthetic DNA
damaging agents.
Acknowledgment. We are grateful for support of this research
by the National Institute of General Medical Sciences (GM-
054996).
Supporting Information Available: Complete ref 4. Experi-
mental procedures for the synthesis of the phosphoramidite
precursors to 11 and 12 and the synthesis of 21 and 22.
Histogram of Fe‚EDTA cleavage of cross-linked 18. ESI-MS
of modified oligonucleotides. HPLC and ESI-MS describing the
formation of 22 from 20. Kinetic plots describing first-order
decay of ISC in the presence of sodium azide. NMR spectra of
previously unreported compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
(38) Leroy, J.-L.; Charretier, E.; Kochoyan, M.; Gue´ron, M. Biochemistry 1988,
27, 8894-8898.
(39) Gue´ron, M.; Leroy, J. Methods Enzymol. 1995, 261, 383.
JA0563657
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