Multinuclear NMR and kinetic analysis of DNA interstrand cross-link formation

Article abstract of DOI:10.1021/ja807845n

Recently, a phenylselenyl-modified thymidine (2) was shown to produce DNA interstrand crosslinks (ICLs) via two mechanisms. Photolysis of 2 generates 5-(2′-deoxyuridinyl)methyl radical (1), the reactive intermediate that results from formal hydrogen atom abstraction from the thymine methyl group. This reactive intermediate reacts with the opposing dA and is the first example of a DNA radical that produces ICLs. Kinetic competition studies support the proposal that the rate-limiting step in ICL formation from 1 involves rotation about the glycosidic bond and that the rate constant for this process is influenced by the flanking sequence. Cross-links also form with the opposing dA when 2 is treated with mild oxidants that result in the formation of an intermediate methide-like species (4). Kinetic experiments reveal that 4 reacts with azide, a model nucleophile, via an S₄2′ pathway. Previous experiments suggested that the same product is produced via 1 or 4 but that the initially formed cross-link rearranges during the enzyme digestion and isolation procedures. In situ product analysis by NMR using synthetic, doubly labeled duplex DNA containing ¹³C-2 and ¹⁵N₁-dA provides definitive evidence that the kinetic ICL products formed via the radical and oxidative pathways are the same and correspond to that arising from formal alkylation of N₁-dA. Furthermore, analysis of the thermodynamic product formed upon rearrangement indicates that the primary product isomerizes via an associative mechanism in DNA.

Full text of DOI:10.1021/ja807845n

Published on Web 12/03/2008
Multinuclear NMR and Kinetic Analysis of DNA Interstrand
Cross-Link Formation
Hui Ding,† Ananya Majumdar,*,‡ Joel R. Tolman,† and Marc M. Greenberg*,†
Department of Chemistry and Biomolecular NMR Center, Johns Hopkins UniVersity,
3400 North Charles Street, Baltimore, Maryland 21218
Received October 4, 2008; E-mail: ananya@jhu.edu; mgreenberg@jhu.edu
Abstract: Recently, a phenylselenyl-modified thymidine (2) was shown to produce DNA interstrand cross-
links (ICLs) via two mechanisms. Photolysis of 2 generates 5-(2′-deoxyuridinyl)methyl radical (1), the reactive
intermediate that results from formal hydrogen atom abstraction from the thymine methyl group. This reactive
intermediate reacts with the opposing dA and is the first example of a DNA radical that produces ICLs.
Kinetic competition studies support the proposal that the rate-limiting step in ICL formation from 1 involves
rotation about the glycosidic bond and that the rate constant for this process is influenced by the flanking
sequence. Cross-links also form with the opposing dA when 2 is treated with mild oxidants that result in
the formation of an intermediate methide-like species (4). Kinetic experiments reveal that 4 reacts with
azide, a model nucleophile, via an SN2′ pathway. Previous experiments suggested that the same product
is produced via 1 or 4 but that the initially formed cross-link rearranges during the enzyme digestion and
isolation procedures. In situ product analysis by NMR using synthetic, doubly labeled duplex DNA containing
13C-2 and 15N1-dA provides definitive evidence that the kinetic ICL products formed via the radical and
oxidative pathways are the same and correspond to that arising from formal alkylation of N1-dA. Furthermore,
analysis of the thermodynamic product formed upon rearrangement indicates that the primary product
isomerizes via an associative mechanism in DNA.
their formation.11-14 Recently, we discovered a process by
Introduction
which the DNA radical 1 generated via formal hydrogen atom
DNA interstrand cross-links (ICLs) are responsible for the
cytotoxicity of a number of antitumor agents.1,2 They are also
produced via endogenous cellular lipid oxidation as well as by
exogenous bis-electrophiles.3-5 The physiological effects of this
DNA lesion family are frequently associated with their ability
to block replication and transcription as well as the difficulty
of their repair.1,6-8 Synthetically induced ICLs are also useful
in a variety of biotechnology applications. These include control
of gene expression in mammalian cells and sequence-specific
recognition of DNA at single-nucleotide resolution.9,10 The
importance of ICLs continues to spur creative approaches for
abstraction from the thymine methyl group cross-links with the
opposing dA in the duplex (Scheme 1).15,16 Although this
transformation was originally detected using photolabile syn-
thetic radical precursors (e.g., 2), the ICL was subsequently
identified in γ-irradiated DNA.17 This was the first characteriza-
tion of an ICL produced in DNA by γ-radiolysis via a radical.
ICLs are also produced from the phenyl selenide 2 under a
variety of oxidative conditions following sigmatropic rearrange-
ment of the selenoxide 3 (Scheme 1).15,18,19 This mild and rapid
method for producing ICLs has been used to develop a method
for detecting single-nucleotide polymorphisms.20 In this ap-
plication, covalent linkage between the biotinylated oligonucle-
otide probe and the target enhances selectivity by permitting
† Department of Chemistry.
‡ Biomolecular NMR Center.
(1) Noll, D. M.; Mason, T. M.; Miller, P. S. Chem. ReV. 2006, 106, 277–
301.
(11) Weng, X.; Ren, L.; Weng, L.; Huang, J.; Zhu, S.; Zhou, X.; Weng, L.
Angew. Chem., Int. Ed. 2007, 46, 8020–8023.
(12) Halila, S.; Velasco, T.; De Clercq, P.; Madder, A. Chem. Commun.
2005, 936–938.
(2) Scha¨rer, O. D. ChemBioChem. 2005, 6, 27–32.
(3) Stone, M. P.; Cho, Y.-J.; Huang, H.; Kim, H.-Y.; Kozekov, I. D.;
Kozekova, A.; Wang, H.; Minko, I. G.; Lloyd, R. S.; Harris, T. M.;
Rizzo, C. J. Acc. Chem. Res. 2008, 41, 793–804.
(4) Kozekov, I. D.; Nechev, L. V.; Moseley, M. S.; Harris, C. M.; Rizzo,
C. J.; Stone, M. P.; Harris, T. M. J. Am. Chem. Soc. 2003, 125, 50–
61.
(13) Alzeer, J.; Schärer, O. D. Nucleic Acids Res. 2006, 34, 4458–4466.
(14) Ali, M. M.; Nagatsugi, F.; Mori, K.; Nagasaki, Y.; Kataoka, K.; Sasaki,
S. Angew. Chem., Int. Ed. 2006, 45, 3136–3140.
(15) Hong, I. S.; Ding, H.; Greenberg, M. M. J. Am. Chem. Soc. 2006,
128, 485–491.
(5) Kim, H.-Y. H.; Voehler, M.; Harris, T. M.; Stone, M. P. J. Am. Chem.
Soc. 2002, 124, 9324–9325.
(16) Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 3692–
3693.
(6) Niedernhofer, L. J.; Lalai, A. S.; Hoeijmakers, J. H. J. Cell 2005, 123,
1191–1198.
(17) Ding, H.; Greenberg, M. M. Chem. Res. Toxicol. 2007, 20, 1623–
1628.
(7) Tomasz, M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73–87.
(8) Smeaton, M. B.; Hlavin, E. M.; McGregor Mason, T.; Noronha, A. M.;
Wilds, C. J.; Miller, P. S. Biochemistry 2008, 47, 9920–9930.
(9) Puri, N.; Majumdar, A.; Cuenoud, B.; Miller, P. S.; Seidman, M. M.
Biochemistry 2004, 43, 1343–1351.
(18) Hong, I. S.; Ding, H.; Greenberg, M. M. J. Am. Chem. Soc. 2006,
128, 2230–2231.
(19) Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 10510–
10511.
(10) Grant, K. B.; Dervan, P. B. Biochemistry 1996, 35, 12313–12319.
(20) Peng, X.; Greenberg, M. M. Nucleic Acids Res. 2008, 36, e31.
9
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A R T I C L E S
Ding et al.
Scheme 1. Cross-Link Formation via Radical and Oxidative Mechanisms
Scheme 2. Alternative Pathways for Cross-Link Formation
stringent washings and enables signal amplification that reduces
the need for PCR amplification. Hydroxyl radical cleavage
experiments revealed that the cross-links produced via the
oxidative pathway also exclusively involve the opposing dA.
Although stability studies suggested that the radical and
oxidative pathways yield the same product 5, direct evidence
for this and the exact structure of the product(s) were lacking.15,18
We now describe one- and two-dimensional NMR experiments
using doubly isotopically labeled (13C, 15N) oligonucleotides that
provide definitive structural evidence for the cross-linked
product. In addition, NMR and kinetic experiments provide
mechanistic details that will be useful in future applications of
the cross-linking chemistry attributable to 2 and related mol-
ecules.21
methylene carbon of the modified thymine and the opposing
N1-dA requires that the pyrimidine adopt the syn conformation.
Previous kinetic competition studies suggested that rotation
about the glycosidic bond was rate-limiting for cross-linking
via the radical.15 For reaction via the longer-lived methide-type
intermediate 4, the rate constant for rotation about the glycosidic
bond is less crucial. In addition, although we favored initial
formation of 5, reaction at C6 of 1 and/or 4 followed by formal
[3,3]-sigmatropic rearrangement of 7 could not be ruled out
under either set of reaction conditions (Scheme 2). Finally, the
potential cross-link products could be formed under oxidative
conditions via SN1, SN2, and/or SN2′ mechanisms. The NMR
and kinetic experiments described below address these questions.
Results and Discussion
The monomeric components (1 and dA) in the dimeric
nucleoside 6 obtained following enzyme digestion of the ICLs
formed under radical and oxidative conditions were bonded to
one another via the C5 methylene of the thymidine derived from
2 and the N6 amino group of dA. It was inconceivable that such
a connectivity in 6 could form directly via a radical reaction
(Scheme 1).16 Inspection of molecular models suggested that
N1-dA is the most likely site of reaction with the radical when
the nucleotide is in its syn conformation. Furthermore, consid-
eration of nitrogen nucleophilicities also pointed to N1 alkylation
by the rearranged selenoxide 4 formed under oxidative
conditions.22,23 We hypothesized that the radical and methide-
type intermediates (1 and 4, respectively) reacted at N1-dA to
produce a kinetic product 5 that rearranged to 6 during the
isolation procedures (Scheme 1). Reaction between the exocyclic
NMR Product Analysis and Mechanism of Isomerization
Using Isotopically Labeled Oligonucleotides. We previously
proposed that syn-1 adds to N1-dA to form 5 as a kinetic product
that ultimately rearranges to the more stable N6-alkylation
product 6 (Scheme 1). Cross-link 5 was also proposed as the
primary product when DNA containing 2 was exposed to
oxidative conditions. Dissociative and associative rearrangement
mechanisms are conceivable (Scheme 3), but kinetic experiments
did not distinguish between them.15 These experiments also did
not eliminate 7 as a possible product. NMR analysis of labeled
oligonucleotides has proven useful in studies of cross-linked
DNA.4,24 We recognized that carrying out the cross-linking
(22) Weinert, E. E.; Frankenfield, K. N.; Rokita, S. E. Chem. Res. Toxicol.
2005, 18, 1364–1370.
(21) Peng, X.; Hong, I. S.; Li, H.; Seidman, M. M.; Greenberg, M. M.
(23) Veldhuyzen, W. F.; Shallop, A. J.; Jones, R. A.; Rokita, S. E. J. Am.
Chem. Soc. 2001, 123, 11126–11132.
J. Am. Chem. Soc. 2008, 130, 10299–10306.
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Mechanistic Analysis of DNA Interstrand Cross-Link Formation
A R T I C L E S
Scheme 3. Structure and Mechanism Elucidation Using Isotopic Labeling
Harris and co-workers26 for synthesizing 15N1-dA, which was
converted to 9 via standard methods. Similarly, 10 was prepared
via a slightly modified version of the route previously used to
synthesize the unlabeled phosphoramidite.16,27 The 13C label was
Scheme 4. Mechanistic Distinction Using Double Isotopic Labeling
reaction using 13C5-2 and 15N1-dA would enable direct detection
of 5 and/or 7 (Schemes 3 and 4). In addition, the two possible
rearrangement mechanisms involving 5 would also be distin-
guishable from one another using the doubly labeled duplex
(Scheme 3). The N1-alkylation product 5 and subsequent
formation of 6b via an associative (Dimroth-like) rearrangement
mechanism would be identifiable via JCN and/or JHN coupling
in the products. In principle, coupling between H2-dA and 15N1-
dA of the purine should be detected in 5, but its presence in 6
depends upon the rearrangement mechanism. If the rearrange-
ment proceeds via a dissociative mechanism to give 6a, the
coupling will be preserved, but it will be lost if an associative
mechanism to give 6b is involved (Scheme 3). The opposite is
true for the JCN coupling, as the connectivity between the labeled
atoms in 5 is maintained during an associative rearrangement.
The doubly labeled substrate also enables us to detect the third
possible cross-linked product 7 and the rearrangement pathway
that results from initial cross-linking between the C6 position
of the modified pyrimidine and N1-dA (Scheme 2). This pathway
would be evident from an absence of significant JCN coupling
in the primary and rearrangement products (Scheme 4) as well
as the distinctive chemical shift of the exocyclic methylene
group.
introduced via transmetalation of the bis(TBDPS) ether of
5-iodo-2′-deoxyuridine with 13CH3I using the procedure of Aso
et al.28 The oligonucleotide in 8 containing 2 was purified by
reversed-phase HPLC following initial purification by denaturing
polyacrylamide gel electrophoresis (PAGE) in order to remove
any oligonucleotide containing 5-hydroxymethyl-2′-deoxyuri-
dine, which is formed from adventitious oxidation of 2. Gel
electrophoresis was sufficient for purification of the oligonucle-
otide containing 15N1-dA.
Initial NMR experiments were carried out using 11 and 12,
the labeled analogues of 2 and dA, respectively. Oxidation
(NaIO4, 100 mM) of the nucleoside mixture (20 mM) in
phosphate buffer (10 mM, pH 7.2) and NaCl (100 mM) followed
by in situ analysis provided a spectrum consistent with 13 (5),
as evidenced by a two-dimensional (2D) heteronuclear single-
quantum correlation (HSQC) experiment that revealed correla-
tion between the H2-dA proton (δ 8.65) and the 15N-labeled N1
These experiments were carried out on a duplex containing
the same sequence as a 16 base pair substrate (8) previously
shown to produce ICLs from 2 under radical and oxidative
conditions and on the corresponding mononucleosides 11 and
12 (Scheme 5).15,16 The 15N1-dA phosphoramidite 9 was
synthesized using procedures reported by Gao and Jones25 and
(25) Gao, X.; Jones, R. A. J. Am. Chem. Soc. 1987, 109, 1275–1278.
(26) Kim, H. H.; Finneman, J. I.; Harris, C. M.; Harris, T. M. Chem. Res.
Toxicol. 2000, 13, 625–637.
(27) Hong, I. S.; Greenberg, M. M. Org. Lett. 2004, 6, 5011–5013.
(28) Aso, M.; Kaneko, T.; Nakamura, M.; Koga, N.; Suemune, H. Chem.
Commun. 2003, 1094–1095.
(24) Young-Jin, C.; et al. J. Am. Chem. Soc. 2005, 127, 17686–17696.
9
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A R T I C L E S
Ding et al.
Scheme 5. Cross-Linking of Isotopically Labeled Monomers
The experiment utilizing isotopically labeled monomeric
components (Figure 1, Scheme 5) proved to be very useful for
subsequent studies in duplex 8. NMR was again used to
characterize the crude mixture of products obtained from
reaction of 8 (1.0 mM) with NaIO4 (5 mM) in potassium
phosphate (10 mM, pH 7.2) and NaCl (100 mM). Consistent
with the experiment in which monomeric molecules were
employed, a resonance was observed in the proton dimension
at δ 7.90, which was correlated with the 15N coupled to the 13
C
(Figure 2A,B). Comparable results were obtained when 8 (1.5
mM) was photolyzed to produce 1, although the signal-to-noise
ratio was not as great because of a lower cross-link yield.31
Following consumption of 4 or photolysis of 2, no signal
consistent with that of 7 (Scheme 4) was detected, as evidenced
by the absence of any 1H signals in the δ 5.0-6.5 region, where
the vinyl protons from the exocyclic methylene group were
expected to resonate.19,21 These experiments provide definitive
evidence for 5 as the primary cross-link product produced from
phenyl selenide 2 under oxidative and radical conditions.
NMR also provided valuable mechanistic information regard-
ing the rearrangement of 5 to 6 within duplex DNA. The crude
cross-linked sample was subjected to the previously employed
purification and isolation conditions16 that gave rise to 6 but
not to piperidine, as was used above for the product obtained
from reaction of the monomers. The resulting HSQC NMR
spectra failed to show any H2-N1 correlations (data not shown).
Since the experiments utilizing the monomeric material proved
that such a coupling was detectable, the absence of any such
coupling in the rearranged polymeric cross-link suggested that
in the duplex, 5 isomerizes via an associative (Dimroth-type)
mechanism to 6b (Scheme 3).32 We also considered the
possibility that the absence of coupling between the 15N6 in 6b
and the adjacent protons or 13C might be due to larger coupling
in the monomeric material than in the duplex. Consequently,
we digested the rearranged cross-link product and purified 14b,
the nucleoside form of 6b. In D2O solvent, the NMR spectra
nitrogen derived from 11 (Figure 1A,B and Scheme 5). The
15N carrier was positioned near 230 ppm, in the characteristic
chemical shift range for aromatic 15N resonances (200-230
ppm), and the HSQC pulse sequence parameters were adjusted
to detect the long-range H2-N1 coupling constant (∼15 Hz).
To detect the JCN coupling constant, two high-resolution, long-
range H2-N1 HSQCs were acquired (JH N ≈ 15 Hz), one in
1
the presence and the other in the absence2 of 13C decoupling in
the 15N (ω1) dimension. In the presence of 13C decoupling
(Figure 1A), the H2-N1 cross-peak appears as a singlet. In the
absence of 13C decoupling (Figure 1B), the H2-N1 cross-peak
splits into a doublet due to the JCN coupling constant. The small
offset between the two components of the N-C doublet in the
1H (ω2) dimension is the consequence of a small, long-range
H2-13C coupling (JH C). Since no 13C decoupling is employed
2
anywhere in the HSQC pulse sequence, the 13C spin states (Cv
and CV) are completely undisturbed throughout the experiment.
This results in the observation of exclusive correlations between
N1 nitrogens and H2 protons associated with Cv spins [N1(Cv)
(ω1) f H2(Cv) (ω2)] and N1 nitrogens and H2 protons associated
with CV spins [N1(CV) (ω1) f H2(CV) (ω2) ], also called an
E.COSY29,30 effect. The displacement between the two peaks
again failed to show an H2-N1 correlation (data not shown).
The absence of coupling between the exocyclic amine in 6b
and the adjacent magnetically active nuclei, which would appear
as H6-N6 cross peaks, is attributed to rapid exchange of the
labile H6 protons with water, especially under non-hydrogen-
bonded conditions. Splittings due to JCN couplings (between the
exocyclic adenine nitrogen and the exocyclic methylene carbon
on the thymine) could not be observed in high-resolution
1H-13C HSQC spectra either, because the cross-peaks were too
broad, indicating intermediate conformational-exchange broad-
ening or conformational heterogeneity in the cross-link region.
Subsequently, we attempted to detect the JCN coupling via
15N-13C splittings in an H6-N6 HSQC correlation spectrum.
Accordingly, the sample was lyophilized and redissolved in 95:5
H2O/D2O acetate buffer (10 mM, pH 5.0). The HSQC param-
eters were adjusted to detect the H6-N6 correlation via the one-
is equal to JH C. Exposure of dimeric nucleoside 13 to piperidine
2
to facilitate rearrangement to 14a (6a) yielded the expected
product, as evidenced by a comparison of the resulting 1H NMR
and HPLC data to those previously reported.16 A H2-N1 HSQC
experiment revealed that the 15N label was still adjacent to H2-
dA in 14a (Figure 1C), indicating that the rearrangement
proceeded at least in part via a dissociative mechanism.
However, we cannot rule out the possibility that a portion of
the reaction proceeds via an associative mechanism for the
following two reasons: (a) the NMR experiments were carried
out in D2O solvent, thus precluding the observation of labile
H6 protons on the exocyclic amine (N6) in 14a; (b) even in H2O
solvent, rapid exchange of the H6 protons may prevent observa-
tion of H6-N6 cross-peaks in the HSQC experiment.
(29) Griesinger, C.; Sørensen, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1985,
107, 6494–6496.
(30) Montelione, G. T.; Wagner, G. J. Am. Chem. Soc. 1989, 111, 5474–
(31) See the Supporting Information.
(32) Fujii, T.; Itaya, T. Heterocycles 1998, 48, 359–390.
5475.
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Mechanistic Analysis of DNA Interstrand Cross-Link Formation
A R T I C L E S
Figure 1. NMR analysis of the reaction of 11 (13C5-2) and 12 (15N1-dA) in the presence of NaIO4. (A, B): 2D H2-N1 HSQC of the crude reaction mixture
acquired at high resolution (0.47 Hz/point) in the 15N dimension in (A) the presence and (B) the absence of 13C decoupling in the 15N dimension. The peaks
marked with * in (A) and (B) belong to unreacted starting material. The contour thresholds are not identical in (A) and (B). (C) 1D HSQC of HPLC-purified
14a following rearrangement of 13 induced by piperidine.
Figure 2. NMR analysis of the reaction of 8 in the presence of NaIO4. (A, B): 2D H2-N1 HSQC of the crude reaction mixture acquired at high resolution
(0.47 Hz/point) in the 15N dimension in (A) the presence and (B) the absence of 13C decoupling in the 15N dimension. Peaks belonging to unreacted starting
material are not shown. The contour thresholds are not identical in (A) and (B).
bond JH N coupling (∼90 Hz), and the 15N carrier was positioned
6
6
at 80 ppm, which is characteristic of the exocyclic N6 resonance
and very distinct from the aromatic N1 resonance (200-230
ppm). A low-resolution HSQC (Figure 3) clearly shows the H6
proton (δ ≈ 7.55 ppm) correlated with a 15N resonance at 83.3
ppm. This unequivocally establishes that the 15N label is at the
exocyclic N6 position (14b), which is positive evidence in
support of the associative mechanism (Scheme 3). Attempts to
detect a JCN coupling by recording high-resolution spectra with
and without 13C decoupling were unsuccessful because the 15
N
resonance was rather broad and decayed rapidly. This observa-
tion is consistent with the similar broadening observed in the
13C resonances in the 1H-13C HSQC spectra, indicating mobility
on an intermediate exchange time scale in the cross-linked
region.
The change in mechanism for the rearrangement of 5 to 6b
in the duplex compared with that for the monomeric system
(13 to 14) is ascribed to differences in reaction conditions. More
pertinent is the question of why the associative rearrangement
mechanism of 5 to 6b in DNA is distinct from the behavior
observed for the o-quinone methides studied by Rokita and
co-workers,22,23,33,34 which react reversibly with DNA via a
Figure 3. NMR analysis of 14b obtained following enzyme digestion of
the cross-linked product produced in the reaction of 8 with NaIO4. 1H-15N
HSQC of the crude reaction mixture in 95:5 H2O/D2O acetate buffer (10
mM, pH 5.0), acquired at low resolution in the 15N dimension (20.0 Hz/
point), showing a cross-peak between the exocyclic H6 proton and N6
nitrogen. The 15N excitation frequency was positioned at 80 ppm, and the
HSQC parameters were optimized to detect the one-bond JHN coupling
constant (90 Hz).
dissociative mechanism (Scheme 6). We speculate that the
dissociative mechanism for 5 is less favorable than for the Rokita
methides because the latter have a relatively acidic proton (the
phenolic H) whose removal drives the reaction toward o-quinone
9
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A R T I C L E S
Ding et al.
allylic alcohol is probably less favorable in this case because it
would produce the unstable (nonaromatic) product, whereas
reaction at the exocyclic methylene in 4 regenerates the aromatic
pyrimidine ring.
Rate-Limiting Glycosidic Bond Rotation in Radical-
Mediated Cross-Link Formation. Formation of 5 requires that
the methide 4 or radical 1 adopt the syn conformation by rotating
about the glycosidic bond. DNA breathing limits the rate at
which rotation occurs. Although the rate constants for base-
pair opening in duplexes containing 2 (or 4) have not been
measured, NMR studies on other duplexes suggest that rate
constants g103 s-1 at 25 °C are possible.39-41 This time scale
is short compared with that on which the methide reacts to form
cross-links but not that for a radical, whose lifetime is inherently
more limited.18 The rate constant for ICL formation (kICL) from
1 was estimated by carrying out competition studies with thiol
(RSH) under anaerobic conditions (eq 1):
Figure 4. Observed rate constant for the formation of 15 upon reaction of
azide with monomeric 4 as a function of azide concentration.
Scheme 6. Dissociative, Reversible Alkylation by an o-Quinone
Methide
k
kX
[ssDNA]
[ICL]
)
RSH[RSH] +
(1)
kICL
kICL
where kRSH is the rate constant for the reaction involving the
thiol. When the radical was flanked on both sides by dC, phenyl
selenide 2 and aryl sulfide 17 were employed separately as
photochemical precursors. In addition, glutathione (GSH) and
ꢀ-mercaptoethanol (BME) were used as thiols. The magnitudes
of the rate constants were affected by the presence of 5-hy-
droxymethyl-2′-deoxyuridine (16) and background levels of
ICLs. Hence, it was imperative that HPLC-purified oligonucle-
regeneration. A similar pathway is not available to the kinetic
cross-link product 5 described in these studies.
Kinetic Determination of Cross-Link Formation Under
Oxidative Conditions. Formation of 5 via the oxidative pathway
that proceeds through 4 could occur via an SN1 or SN2′ pathway.
These were distinguished from one another by using UV
absorption spectroscopy at 25 °C to measure the rate constant
for the reaction of azide with monomeric 4 produced by
oxidation of 11. Absorption spectroscopy was a convenient tool
for following this reaction because the methide 4 absorbs weakly
in the 260-270 nm region where the maxima for nucleosides
are typically observed. Hence, the absorption increase at 270
nm was used to measure formation of the product 15. Azide
otides from which oligonucleotides containing 16 had been
removed as well as freshly hybridized duplexes be used for these
experiments. Failure to remove the impurities gave rise to
artificially high calculated values for kICL. The rate constants
for ICL formation determined using 17 and 2 were indistin-
guishable from each another (Table 1) and were a factor of ∼4
smaller than that reported previously using oligonucleotides that
were not HPLC-purified.15 Furthermore, if one assumes that
BME and GSH react with 5-(2′-deoxyuridinyl)methyl radical 1
with equal rate constants, one obtains kICL values that are within
experimental error of one another.42
Altering the flanking sequence has a more significant effect
on ICL yields, as measured by phosphorimage analysis of 32P-
labeled substrates separated by denaturing PAGE. The cross-
link yields in the absence of thiol (Table 1) were greater when
5-(2′-deoxyuridinyl)methyl radical 1 was flanked by pyrimidines
than by purines. We attribute this to more efficient intrastrand
was used as a nucleophile instead of dA in order to avoid
interference with the absorption spectrum of the product. This
was particularly important since the nucleophile was present in
large excess in order to fulfill the requirements of pseudo-first-
order reaction conditions. The observed rate constant for the
reaction of azide with monomeric 4 to produce 15 exhibited a
linear dependence on nucleophile concentration (Figure 4) and
is consistent with the SN2′ mechanism. These data yield a
bimolecular rate constant of 1.9 × 10-2 M-1 s-1 for the reaction
of monomeric 4 with azide. Extrapolation to the reaction
between the 4 and dA in a duplex suggests that cross-link
formation under oxidative conditions also occurs via an SN2′
pathway. Attack by the nucleophile on the carbon of the allylic
phenyl selenate 4 is atypical for this molecular family.35-38
However, the more typical attack on selenium to produce the
(35) Reich, H. J.; Yelm, K. E.; Wollowitz, S. J. Am. Chem. Soc. 1983,
105, 2503–2504.
(36) Bourland, T. C.; Carter, R. G.; Yokochi, A. F. T. Org. Biomol. Chem.
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17986 J. AM. CHEM. SOC. VOL. 130, NO. 52, 2008

Mechanistic Analysis of DNA Interstrand Cross-Link Formation
A R T I C L E S
Table 1. Effect of the Flanking Sequence on ICL Formation from
5-(2′-Deoxyuridinyl)methyl Radical 1
rate constants were uncorrelated. ICL formation was the fastest
when the 1 was flanked by dC, and it was not possible to
accurately measure kICL when dA was the flanking nucleotide
because the yield was already too low in the absence of thiol
to accurately measure the change in the product as a function
of thiol concentration. The reactivity of 5-(2′-deoxyuridinyl-
)methyl radical 1 was in contrast to that of 4, whose rate
constants for ICL formation were greater when flanked by
pyrimidines but produced comparable yields of cross-links in
all sequences.20 Regardless, the most significant observation is
that kICL is within the range that one would expect for the rate
constant for nucleotide flipping and consistent with the previous
proposal that isomerization of the radical into the syn conforma-
tion is the rate-determining step in ICL formation from 1.15,39-41
Conclusions
NMR analyses of synthetic oligonucleotides containing 13C
and 15N labels have provided definitive proof for the kinetic
product 5 formed by a novel DNA radical cross-linking reaction.
The NMR experiments also prove that the same product is
obtained via an oxidative pathway and that the initially formed
product isomerizes to the thermodynamic cross-link 6b via an
associative mechanism in duplex DNA. The mechanism of the
rearrangement is distinct from that involving o-quinone methides
and suggests that the cross-links produced via 2 are chemically
robust.34 The irreversible nature of this cross-linking reaction
suggests that it will be useful for kinetically trapping molecules
in its vicinity. In this regard, kinetic experiments suggest that
radical reactions will be influenced by the rate constant for
isomerization about the glycosidic bond and that the reactivity
of the methide-like species 4 is controlled by the rearomatization
driving force. Knowledge of the mechanistic aspects of this
chemistry will be useful in developing applications of the phenyl
selenide precursor 2 as well as in the design of molecules with
similar reactivity.20,21
a Each rate constant is the average of at least two experiments, each
carried out in triplicate. b For the reaction carried out in the absence of
thiol. Each value is the average of at least three measurements.
cross-link formation when 1 is flanked by purines. Addition of
1 (and the analogous radical 23 generated from 5-methyl-2′-
deoxycytidine) to the C8 position of dG has been well-studied,
and the subsequently formed tandem lesions are biologically
significant.43-45 In contrast, tandem lesions resulting from
addition of 1 or 23 to an adjacent pyrimidine have been observed
less frequently and are not as well studied.46,47 Although it is
unknown if this is due to a smaller rate constant for addition
by the radicals to pyrimidines than to purines, such a scenario
is consistent with the phenomenological observations reported
in the literature. The maximum ICL yields and cross-linking
Acknowledgment. We are grateful for support of this research
by the National Institute of General Medical Sciences (GM-054996
to M.M.G. and GM-075310 to J.R.T.) and the NSF (MCB-0615786
to J.R.T.). M.M.G. thanks Professor Ned Porter for a helpful
discussion that provided the impetus for the NMR experiments.
Supporting Information Available: Complete description of
experimental procedures, NMR spectra of 9 and 10, HSQC
spectra of the cross-link product formed from photolysis of 8,
and complete ref 24. This material is available free of charge
via the Internet at http://pubs.acs.org.
(43) Romieu, A.; Bellon, S.; Gasparutto, D.; Cadet, J. Org. Lett. 2000, 2,
1085–1088.
(44) Zhang, Q.; Wang, Y. Nucleic Acids Res. 2005, 33, 1593–1603.
(45) Jiang, Y.; Hong, H.; Cao, H.; Wang, Y. Biochemistry 2007, 46, 12757–
12763.
(46) Friedel, M. G.; Pieck, J. C.; Klages, J.; Dauth, C.; Kessler, H.; Carell,
T. Chem.sEur. J. 2006, 12, 6081–6094.
(47) Cheek, J.; Broderick, J. B. J. Am. Chem. Soc. 2002, 124, 2860–2861.
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