DNA interstrand cross-link formation by the 1,4-dioxobutane abasic lesion

Article abstract of DOI:10.1021/ja9061695

The oxidized abasic lesion 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly with a single-strand break by a variety of DNA-damaging agents that abstract a hydrogen atom from the C5′-position. Independent generation of the DOB lesion in DN

Full text of DOI:10.1021/ja9061695

Published on Web 10/06/2009
DNA Interstrand Cross-Link Formation by the 1,4-Dioxobutane
Abasic Lesion
Lirui Guan and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles Street,
Baltimore, Maryland 21218
Received July 23, 2009; E-mail: mgreenberg@jhu.edu
Abstract: The oxidized abasic lesion 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly
with a single-strand break by a variety of DNA-damaging agents that abstract a hydrogen atom from the
C5′-position. Independent generation of the DOB lesion in DNA reveals that it reversibly forms interstrand
cross-links (ICLs) selectively with a dA opposite the 3′-adjacent nucleotide. Product studies and the use of
monoaldehyde models suggest that ICL formation involves condensation of the dialdehyde with the exocyclic
amine. Mechanistic studies and inspection of molecular models indicate that the local DNA environment
and proximity of the exocyclic amine determine the selectivity for reaction with dA. Proximity control of the
electrophile’s reactivity is distinct from that of structurally similar freely diffusing molecules. ICL formation
by a DOB lesion that is adjacent to a single-strand break is potentially significant because the product
constitutes a “clustered” or “complex” lesion. Clustered lesions can lead to highly deleterious double-strand
breaks upon nucleotide excision repair.
antitumor agents form interstrand cross-links.9-12 The latter
produces high yields of interstrand cross-links (ICLs) in a
Introduction
DNA is the cytotoxic target of a number of therapeutic agents
and is also at the root of carcinogenesis. The chemical basis of
these biological effects is not clear in many situations because
a myriad of products are formed when DNA is exposed to
various endogenous and exogenous damaging agents.1 In
addition to single- and double-strand breaks, some agents
produce interstrand cross-links, which are an important family
of lesions because they are absolute blocks of replication and
transcription.2,3 By far the largest class of DNA lesions are those
that result from modification of the sugar or nucleobase of an
individual nucleotide. More than 50 modified nucleotides have
been characterized.4,5 Many of these lesions decrease the fidelity
of polymerases, resulting in mutations. However, a small number
exhibit unusual chemical reactivity that may have significant
biological consequences. For instance, two lesions have been
discovered to irreversibly inhibit one or more of the DNA repair
enzymes that have evolved to excise damage from the
biopolymer.6-8 In two other examples, it was observed that the
abasic site (AP) formed by formal hydrolysis of the nucleotide’s
glycosidic bond and the C4′-oxidized abasic site (C4-AP) that
is produced by ionizing radiation, bleomycin, and several other
sequence-dependent manner. We report that the oxidized abasic
site dioxobutane (DOB) also forms interstrand cross-links and
that its nucleotide selectivity and chemistry are distinct from
those reported for AP and C4-AP.
5′-(2-Phosphoryl-1,4-dioxobutane) (DOB) is a product of
DNA oxidation.13,14 Although the detailed mechanism of its
formation is not understood, it is believed to arise from C5′-
oxidation under O2 -deficient conditions (Scheme 1).15,16 DOB
is unique among oxidized abasic lesions in that its formation is
accompanied by a strand break at the adjacent nucleotide.
Consequently, DNA damage containing DOB constitutes a
(9) Dutta, S.; Chowdhury, G.; Gates, K. S. J. Am. Chem. Soc. 2007, 129,
1852–1853.
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J.-L. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14032–14037.
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2008, 130, 9646–9647.
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301.
(3) Scha¨rer, O. D. ChemBioChem 2005, 6, 27–32.
(4) Cadet, J.; Douki, T.; Gasparutto, D.; Ravanat, J. L. Mutat. Res. 2003,
531, 5–23.
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J. Am. Chem. Soc. 2009, 131, 11132–11139.
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Radical Biol. Med. 2002, 32, 1102–1115.
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Kool, E. T., Ed.; Elsevier: Amsterdam, 1999; Vol. 7, pp 553-592.
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2004, 17, 1406–1413.
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J. AM. CHEM. SOC. 2009, 131, 15225–15231 15225

A R T I C L E S
Guan and Greenberg
Scheme 1
Scheme 2
tandem lesion, which is defined as two contiguously damaged
nucleotides. Tandem lesions are a subset of clustered (or
complex) lesions, which are defined as two damage sites within
1-2 helical turns. Clustered lesions, particularly those in which
the lesions are in close proximity to one another, adversely affect
DNA repair.17-21 They were even postulated to lead to double-
strand breaks during misrepair.22 Recently, this was unambigu-
ously demonstrated during nucleotide excision repair of an
interstrand cross-link adjacent to a single-strand break that
resulted from the chemical promiscuity of C4-AP.23
Scheme 3
The structure of DOB boded well for the likelihood that it
too may be very reactive. For instance, the presence of the
ꢀ-phosphate suggested that it is a potential source of 2-butene-
1,4-dial, a product of furan metabolism, which alkylates
DNA.24-27 Independent generation of DOB in oligonucleotides
from a photochemical precursor revealed that the 1,4-dicarbonyl
compound forms a stable cyclic adduct with Tris, and served
as the basis of a sensitive method for its selective detection.28,29
The structurally related 1,4-dicarbonyl-containing lesion C4-
AP reacts readily with simple amines and dA and dC in duplex
DNA.11,12,30,31 Hence, it seemed plausible that DOB would also
react with DNA nucleobases.
Results and Discussion
Oligonucleotides containing the photolabile DOB precursor
were prepared as previously described from 1 (Scheme 2).28
Following hybridization with the template and flanking oligo-
nucleotides to form the ternary complexes, the chemically labile
DOB lesion was generated as needed via photolysis (60 min)
in a Rayonet photoreactor at λmax ) 350 nm from the bis-o-
nitrobenzyl precursor. Hybridization was carried out prior to
photochemical generation of the oxidized abasic site to minimize
adventitious cleavage of the DOB lesion. The flanking sequence
was varied (2a-t, Scheme 3) to determine the effect of local
DNA structure on DOB’s reactivity with each of the native
nucleotides that contain one or more nucleophilic nitrogen
atoms. Native gel analysis showed that the ternary complexes
containing DOB produced upon irradiation of the precursor
(Scheme 2) remained hybridized under the subsequent incuba-
tion conditions (data not shown).
Preferential Interstrand Cross-Linking by DOB with dA14.
Examination of molecular models and prior studies on the
reactivity of AP and C4-AP suggested that dN14 of the ternary
complex would be the preferred reaction position.9,11,12 Con-
sequently, we examined the reactivity of DOB in a series of
ternary complexes in which unreactive thymidine was incor-
porated at positions dN15 and dN16 while the nucleotide was
varied at dN14 (Table 1). Incubation in phosphate-buffered (10
mM, pH 7.2) saline (100 mM) at 37 °C for 24 h yielded >30%
ICL (3) when dA was present at position 14, but less than 1%
when any other nucleotide was incorporated at this site. In the
two instances (2a, 2c) in which ICLs were detected hydroxyl
radical cleavage experiments revealed that cross-linking occurred
exclusively at dN14.32,33 The lack of reaction with nebularine
(Ne) was an initial indication that the exocyclic amine of dA
was involved in cross-linking. This is similar to what was
observed in the cross-linking reactions of C4-AP.11,12 As
(17) Georgakilas, A. Mol. BioSyst. 2008, 4, 30–35.
(18) Kozmin, S. G.; Sedletska, Y.; Reynaud-Angelin, A.; Gasparutto, D.;
Sage, E. Nucleic Acids Res. 2009, 37, 1767–1777.
(19) Paap, B.; Wilson, I., D. M.; Sutherland, B. M. Nucleic Acids Res.
2008, 36, 2717–2727.
(20) Lomax, M. E.; Gulston, M. K.; O’Neill, P. Radiat. Prot. Dosim. 2002,
99, 63–68.
(21) Blaisdell, J. O.; Wallace, S. S. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 7426–7430.
(22) Gulston, M.; de Lara, C.; Jenner, T.; Davis, E.; O’Neill, P. Nucleic
Acids Res. 2004, 32, 1602–1609.
(23) Sczepanski, J.; Jacobs, A. C.; Van Houten, B.; Greenberg, M. M.
Biochemistry 2009, 7565–7567.
(24) Chen, B.; Vu, C. C.; Byrns, M. C.; Dedon, P. C.; Peterson, L. A.
Chem. Res. Toxicol. 2006, 19, 982–985.
(25) Gingipalli, L.; Dedon, P. C. J. Am. Chem. Soc. 2001, 123, 2664–
2665.
(26) Byrns, M. C.; Vu, C. C.; Neidigh, J. W.; Abad, J.-L.; Jones, R. A.;
Peterson, L. A. Chem. Res. Toxicol. 2006, 19, 414–420.
(27) Byrns, M. C.; Predecki, D. P.; Peterson, L. A. Chem. Res. Toxicol.
2002, 15, 373–379.
(28) Kodama, T.; Greenberg, M. M. J. Org. Chem. 2005, 70, 9916–9924.
(29) Dhar, S.; Kodama, T.; Greenberg, M. M. J. Am. Chem. Soc. 2007,
129, 8702–8703.
(30) Aso, M.; Usui, K.; Fukuda, M.; Kakihara, Y.; Goromaru, T.; Suemune,
H. Org. Lett. 2006, 15, 3183–3186.
(32) See the Supporting Information.
(33) Millard, J. T.; Weidner, M. F.; Kirchner, J. J.; Ribeiro, S.; Hopkins,
P. B. Nucleic Acids Res. 1991, 19, 1885–1892.
(31) Usiv, K.; Aso, M.; Fukuda, M.; Suemune, H. J. Org. Chem. 2008,
73, 241–248.
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DNA Interstrand Cross-Link Formation
A R T I C L E S
Table 1. Interstrand Cross-Link Formation at dN14 (3)
a Yields are the average of at least three replicates.
Table 2. Interstrand Cross-Linking at the Nucleotide Opposite
Table 3. Selective Cross-Linking of DOB at dA14
DOB (N15
)
a Yields are the average of at least three replicates.
a Yields are the average of at least three replicates.
Table 4. Attempted Cross-Linking of DOB at Nucleotides Other
Than dA
discussed below, the lack of reactivity with Ne provides insight
into the structure of the ICL obtained from DOB.
Reactivities at dN15 and dN16 of the ternary complexes
containing DOB were probed in a similar manner. Although
no cross-link products were observed when dA (2q), dC (2r),
dG (2s), or dT (2b) was incorporated at dN16 (data not shown),
modest yields of cross-links were observed (Table 2) with an
opposing dA (2f). However, positioning dC or dG at dN15
yielded less than 1% of the corresponding cross-link product.
The preference for DOB cross-linking with dA14 was further
illustrated by examining the effects of the local sequence on
the cross-linking site (Table 3). The ICL yield varied between
∼22% and 38%, but hydroxyl radical cleavage revealed that
cross-linking occurred exclusively at dA14 in all instances, even
when four contiguous dA nucleotides were present (2j, Table
3).32 The yields of DOB cross-links are comparable to those
involving C4-AP and considerably greater than those of ICLs
resulting from reaction of AP.9,11,12
The preference for DOB cross-linking with dA14 was evident
even when the oxidized abasic site was presented with alternate
nucleotides in this position and others (Table 4). ICL yields
greater than a few percent were obtained only when dA was
present at dN14. Specifically, reaction was only observed with
dC (and in low yield) when this nucleotide was present at dN15
(2g, Table 2). In addition, the two instances in which cross-
linking was detected at dG14 (2c, Table 1) and dG15 (2h, Table
2) occurred in <1% yield.
a Yields are the average of at least three replicates. nd ) not determined.
NaCNBH3. In these respects, the cross-link was similar to the
ICL produced from C4-AP in which the lesion-containing strand
was uncleaved, but distinct from that isolated from the reaction
of an AP site with dG.9,11,12 The ICL containing an uncleaved
C4-AP strand also only forms when a dA is opposite a
3′-adjacent thymidine. These similarities suggested that the
structure of the DOB-dA cross-link might involve condensation
with the N6-amino group of dA, which is how C4-AP reacts
with this nucleotide.12 The lack of reaction with Ne is consis-
tent with this proposal. Although the ICL decomposition kinetics
Structural Characterization of the Interstrand Cross-Link
Product Formed between DOB and dA. The ICL proved to be
chemically labile and was not stabilized by reaction with
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Guan and Greenberg
Scheme 4a
Scheme 7a
a Key: (a) NaH, BnBr, THF; (b) OsO4, NMO, acetone/H2O; (c)
(TBDMS)Cl, imidazole, DMF; (d) Pd(OH)2/C, H2, EtOAc; (e) cyanoethyl
N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2.
a Key: (a) 1,4-butanedial, phosphate buffer (50 mM, pH 7.2); (b)
NaCNBH3, H2O, AcOH (pH 5.0); (c) t-BuMe2SiCl, imidazole, DMF.
Scheme 8a
Scheme 5
a Key: (a) (TBDMS)Cl, imidazole, DMF; (b) Pd(OH)2/C, H2, EtOAc;
(c) cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2.
are discussed below, the cross-link product was sufficiently
stable to enable its isolation and analysis by ESI-MS.32 ESI-
MS of the product obtained from 2k showed that it resulted
from condensation with the complement without loss of water.
Additional information regarding the structure of the cross-link
was obtained by reacting monomeric dA with 1,4-butanedial
as a model for DOB in the polymer.34 Adduct 4 was isolated
by reversed-phase HPLC as a mixture of diastereomers that were
contaminated with dA due to decomposition of the product
during concentration. 1H NMR analysis was difficult due to the
presence of multiple diastereomers and dA. However, MS data
were consistent with the proposed structure.32 Further support
for this assignment was obtained by converting 4 to previously
reported 5 (Scheme 4).35 On the basis of the lability of the ICL
and the data obtained from the DNA and monomer, we propose
that the cross-link product reported in Tables 1-4 results from
condensation of the DOB lesion with the N6-amino group of
dA (6, Scheme 5) in the opposing strand and may exist as an
equilibrium mixture.
Additional inferential support for the biscondensation product
6 was obtained by examining the ability of oligonucleotides
containing model compound 7 or 8 to form cross-links. These
model compounds position an individual carbonyl group in
approximately the same location as each aldehyde from DOB.
Oligonucleotides containing these monoaldehydes were prepared
via solid-phase oligonucleotide synthesis from the protected
vicinal diol phosphoramidites 9 and 10 (Scheme 6).
Table 5. Interstrand Cross-Link Formation by DOB Model
Compounds
a Yields are the average of at least three replicates.
analogue 7 started from homoallylic alcohol 11 (Scheme 7). DNA
containing 8 was prepared from previously reported 12 (Scheme
8), which was obtained from ascorbic acid.38-40 The modified
phosphoramidites were coupled using standard reagents, but longer
coupling times were employed.32 The vicinal diol containing
oligonucleotides were deprotected via standard methods and
purified by denaturing polyacrylamide gel electrophoresis.32
The reactivity of each model compound was examined in
two sequence contexts, each of which contained a dA at dN14
(Table 5). No cross-linking was detected in 13a,b, and less than
1% was observed in 14a,b. These are far less than the 32.6%
and 38.8% ICL yields measured for the analogous sequences
(2a, 2j) containing DOB. These results indicate that ICL
formation by DOB with dA does not involve Schiff base
formation from a single carbonyl group, as is observed for cross-
linking by AP with dG.9
Interstrand Cross-Link Formation between DOB and dA
Is Reversible. It was mentioned above that the ICL from DOB
is unstable. Studies on the reaction of dA with 1,4-butanedial
suggested that the cross-linking should be reversible. However,
other decomposition pathways for the ICL were possible. One
or more of these could result in release of a 5′-phosphate-
containing oligonucleotide and a template that may be alkylated
by the remnants of the DOB lesion. The outcome of such an
alkylation would be identical to reaction of DNA with butene-
Sodium periodate treatment of the oligonucleotides containing
vicinal diols was used to release the labile aldehydes 7 and 8
immediately prior to their use.28,36,37 The respective phosphora-
midites were synthesized using routes (Schemes 7 and 8) that
started from a known substrate. The proper configuration at the
ultimate stereogenic center in 7 and 8 is incorporated in these
starting materials. For instance, the synthesis of the C1-aldehyde
Scheme 6
(34) Mueller, R.; et al. J. Med. Chem. 2004, 47, 5183–5197.
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DNA Interstrand Cross-Link Formation
A R T I C L E S
s-1). Furthermore, the dialdehyde reacted with dC (kdC ) 9.3
× 10-5 s-1) almost 10 times faster than it did with dA. This
apparent discrepancy between reaction of the monomers with
the model compound and that of DOB within the DNA was
mitigated by measuring the decomposition rate constants for
Scheme 9
dC (k-dC ) 7.6 × 10-5 s-1) and dA (k-dA ) 9.2 × 10-6 s-1
)
adducts.32 Perhaps coincidentally, the equilibrium for the model
reaction with dA (Keq ) 1.3) is very similar to that for cross-
linking between dA and DOB in 2i (Keq ) 1.4). Overall, these
data suggest that the inherent reactivity of dC (Keq ) 1.2) and
dA (Keq ) 1.3) with 1,4-butanedial are quite similar, and that
the equilibria for adduct formation with the model compound
are almost identical. These data suggest that, unlike o-quinone
methides, reaction of 1,4-butanedial, and by inference DOB,
with dA is not thermodynamically favored.48,49
1,4-dial, a known metabolite of furan.15,24-27,41 This was
investigated by following the decomposition of isolated 3i.
Because DOB undergoes partial elimination during denaturing
polyacrylamide gel electrophoresis to produce DNA containing
5′-phosphate termini, it was necessary to treat the samples in
such a way so as to enable one to distinguish between this
artifact and direct formation of oligonucleotides containing the
phosphate end group from the ICLs. Consequently, the ICL was
incubated in Tris buffer to capture released DOB as the
previously characterized adduct (Scheme 9).28,29 In addition,
aliquots removed from a sample of 3′-32P-3i as a function of
time were quenched with NaBH4, which converts any remaining
ICL to DOB and stabilizes the latter by reducing it to the diol.
By employing these chemical treatments, it was assumed that
any oligonucleotide containing 5′-phosphate formed during
incubation of 3′-32P-3i would result from ICL decomposition
that does not involve formation of DOB. None of the phosphate
product was detected by denaturing polyacrylamide gel elec-
trophoresis, indicating that reversion to DOB was the sole
pathway for ICL decomposition (data not shown).
The rate constant for reversion of the ICL (3i) to DOB was
initially determined by following its disappearance to less than
50% conversion and treating the decomposition as an irreversible
process. Fitting the decomposition to a first-order decay yielded
a rate constant for its decomposition, k-1 ) 1.7 × 10-5 s-1 (t1/2
) 11.2 h) under these buffer conditions (10 mM phosphate,
pH 7.2).32 A more elaborate analysis was carried out by monitoring
the growth of the ICL (3i), the DOB elimination product (“phos-
phate”, eq 1), and DOB disappearance (2i, Figure 1). The
concentrations of the three species as a function of time were fit
to a kinetic scheme (eq 1) in which ICL formation (k1) and
elimination (k2) are in competition with one another, and cross-
link formation is reversible. The rate constants for each step were
determined using an iterative fitting procedure.42 The rate constant
determined for ICL decomposition using this method is only
slightly higher (k-1 ) 1.9 × 10-5 s-1) than that calculated above.
The fitting procedure also determines that the rate constant for ICL
formation, k1 ) 2.6 × 10-5 s-1, is only slightly faster than its
decomposition, but that DOB undergoes elimination ∼5 times more
slowly (k2 ) 5.6 × 10-6 s-1). The DOB decomposition rate
constant is ∼3-fold slower than that originally reported, but was
measured at 5-fold lower buffer concentration.28
Since the inherent reactivity of the nucleosides did not explain
the preferential cross-linking to dA in DNA, we considered
whether proximity within the ternary complex could provide
an explanation. Molecular models of three sequences were
examined (Figure 3). The models were constructed from B-form
DNA using Spartan. The DOB lesion was introduced by deleting
the corresponding nucleotide without altering the positions of
any of the remaining atoms. The models suggest that the N2-
amino group of dG16 is the closest of the exocyclic amines to
the DOB aldehyde, consistent with modeling of AP-containing
DNA.9 However, cross-linking is not detected at this nucleotide
position (e.g., see ternary complex 2s), which suggests that the
lack of reaction with dG in DNA correlates with the respective
nucleoside’s reduced reactivity with butanedial (Figure 2). The
molecular models do provide a possible explanation for why
reaction is not observed with dC in any of the ternary complexes.
The shortest distance between DOB and the respective exocyclic
amines of dC and dA occurs with the nucleotides at the dN14
position. However, the N6-amine of dA14 is ∼1 Å closer to the
Figure 1. Kinetic fitting of DOB reactivity as a function of time.
k2
k1
phosphate r DOB (2) h ICL (3)
(1)
k-1
Why Is DOB Cross-Linking to dA Preferred? DOB is an
example of a biselectrophile. Other biselectrophiles, including
C4-AP, do not show such selectivity for dA.11,12 It is common
for biselectrophiles to form adducts with dA, dC, and
dG.24,25,43-47 We sought to gain some insight into why DOB
exhibits a preference for forming cross-links with dA by
examining the reactions of the respective nucleosides with the
DOB model compound 1,4-butanedial (Scheme 4). Measuring
the adduct growth rate (Figure 2) did not resolve the issue.
Adduct formation between dG (kdG ) 9.7 × 10-6 s-1) and 1,4-
butanedial was almost as rapid as with dA (kdA ) 1.2 × 10-5
Figure 2. 1,4-Butanedial adduct formation with nucleosides as a function
of time.
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A R T I C L E S
Guan and Greenberg
formation. Anecdotal evidence in support of the hypothesis that
cross-links involving dN15 are less stable is gleaned from the
characterization of 3p by hydroxyl radical cleavage.32 The data
are difficult to interpret because the ICL is much less stable
compared to those involving cross-linking at dN14.
Conclusions
5′-(2-Phosphoryl-1,4-dioxobutane) is an unusual DNA lesion
that is produced by a variety of damaging agents.13,14 It is
atypical in that it is formed concomitantly with a single-strand
break and is itself a biselectrophile. Single-strand breaks are
deleterious to cells. Furthermore, biselectrophiles exhibit a rich
chemistry that is derived from their ability to produce alkylated
lesions that are often mutagenic. Previously, it was shown that
AP and C4-AP abasic lesions react with nucleotides on the
opposing strand to form interstrand cross-links.9,11,12 The latter
forms two types of ICLs, one of which also contains a single-
strand break on the strand that originally contained the lesion.
This “complex” lesion poses a significant challenge for DNA
nucleotide excision repair, resulting is the formation of ex-
tremely deleterious double-strand breaks.23
The DOB lesion exhibits reactivity that is in many respects
similar to that of C4-AP, including high yields of interstrand
cross-links.11,12 Interestingly, cross-links are not observed with the
3′-terminal nucleotide dN45 in the strand that flanks the DOB lesion.
The cross-linked product forms reversibly and predominantly with
dA, as does the ICL obtained from C4-AP in which the lesion-
containing strand is uncleaved. High yields of the cross-links are
obtained despite the reversibility because other reactions do not
rapidly consume the DOB lesion. For instance, DOB does not
readily form ICLs at other positions in the ternary complex, and
elimination is slow compared to cross-link formation. The lability
of the cross-links and the inability to stabilize them with reagents
such as NaCNBH3 presents a challenge for their structural
characterization and detection in cells. It may be possible to
overcome the former by synthesizing a stable model cross-link.
Structural characterization of the DOB cross-links will be particu-
larly interesting if the precedent set by the respective C4-AP cross-
link is repeated.23 On the basis of the nucleotide excision repair of
C4-AP cross-links, those formed by DOB (3) may also be
especially deleterious because their misrepair could give rise to
double-strand breaks. Furthermore, the facile reaction of the 1,4-
dicarbonyl-containing DOB lesion with even the primary aromatic
amine of adenine suggests that this molecule may readily form
cross-links with DNA-binding proteins. We hope to investigate
these issues in due course.
Figure 3. Proximity of potential nucleophilic partners for DOB: (A) 5′-
dT-DOB-dT/5′-(dA)3; (B) 5′-dG-DOB-dG/5′-(dC)3; (C) 5′-dC-DOB-dC/5′-
(dG)3. The models are obtained using Spartan.
DOB aldehyde than is the N4-amine of dC14. The relatively
closer proximity of the exocyclic amine of dA to the DOB
carbonyl compared to that of dC is evident at dN15 and dN16 as
well. Hence, we posit that the selectivity of DOB cross-linking
is governed by its proximity to the respective nucleophiles of
dC and dA in the ternary complexes. Closer proximity may
increase the rate of ICL formation, but it also may correlate
with cross-link stability. Although structural information is not
available, the closer reactants are to one another, the less distorted
and destabilizing the product DNA may be. One should note that
these models suggest that the exocyclic amine in the opposing
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DNA Interstrand Cross-Link Formation
A R T I C L E S
Acknowledgment. We are grateful for support of this research
by the National Institute of General Medical Sciences (Grant GM-
063028). We are grateful to Professor Jack Norton for assistance with
analyzing the kinetics for cross-link formation and Dr. Shanta Dhar
for carrying out preliminary experiments. We thank Aaron Jacobs and
Jonathan Sczepanski for their comments on the manuscript.
Supporting Information Available: Complete description of
experimental procedures, spectral data of new compounds, ESI-
MS spectra of modified oligonucleotides, sample autoradiograms
of hydroxyl radical cleavage experiments, kinetic plots, and
complete ref 34. This material is available free of charge via
the Internet at http://pubs.acs.org.
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