An oxidized abasic lesion as an intramolecular source of DNA adducts

Article abstract of DOI:10.1071/CH10420

5′-(2-Phosphoryl-1,4-dioxobutane) (DOB) is a lesion produced in DNA via a variety of damaging agents. The DOB lesion spontaneously generates cis- and trans-but-2-en-1,4-dial (1) via β-elimination. Cis- and trans-but-2-en-1,4-dial forms exocyclic adducts with nucleosides. We used chemically synthesized DNA containing tritiated DOB incorporated at defined sites to examine the reactivity of cis- and trans-but-2-en-1,4-dial. Although the local DNA sequence does not appear to influence the distribution of nucleoside adducts, we find that DOB generates relatively high yields of cis- and trans-but-2-en-1,4-dial nucleoside adducts that likely are promutagenic.

Full text of DOI:10.1071/CH10420

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2011, 64, 438–442
www.publish.csiro.au/journals/ajc
An Oxidized Abasic Lesion as an Intramolecular Source
of DNA Adducts
A
A B
,
Lirui Guan and Marc M. Greenberg
A
Department of Chemistry, Johns Hopkins University, 3400 N Charles Street,
Baltimore, MD 21218, USA.
B
Corresponding author. Email: mgreenberg@jhu.edu
50-(2-Phosphoryl-1,4-dioxobutane) (DOB) is a lesion produced in DNA via a variety of damaging agents. The DOB lesion
spontaneously generates cis- and trans-but-2-en-1,4-dial (1) via b-elimination. Cis- and trans-but-2-en-1,4-dial forms
exocyclic adducts with nucleosides. We used chemically synthesized DNA containing tritiated DOB incorporated at
defined sites to examine the reactivity of cis- and trans-but-2-en-1,4-dial. Although the local DNA sequence does not
appear to influence the distribution of nucleoside adducts, we find that DOB generates relatively high yields of cis- and
trans-but-2-en-1,4-dial nucleoside adducts that likely are promutagenic.
Manuscript received: 19 November 2010.
Manuscript accepted: 18 December 2010.
The DNA nucleobases readily react with electrophiles and the
products of such reactions can be detrimental or useful. Reac-
tions with electrophiles are exploited for analyzing nucleic
acid structure, such as the selective methylation of purines by
dimethyl sulfate.[1] Other alkylating agents, such as chlor-
ambucil and mitomycin, which form cross-links, are useful as
therapeutic agents.[2,3] A variety of bis-electrophiles form cyclic
adducts with adenine, cytosine and guanine.[4] Some of these
damaging agents such as 4-hydroxy-non-2-en-1-al, a product of
lipid peroxidation, are produced endogenously.[5] Exogenous
agents such as acrolein and other a,b-unsaturated aldehydes
exhibit similar reactivity. Several of these cyclic adducts have
been shown to be promutagenic, indicating a possible role in
carcinogenesis for the agents that produce them.[6] We wish to
report how the generation of a bis-electrophile, trans-but-2-en-
1,4-dial (1) from a DNA lesion affects its ability to form exo-
cyclic adducts.
(dA), 20-deoxycytidine (dC), and 20-deoxyguanosine (dG)
(2–4, Scheme 1).[7–10] The free nucleosides react in the order
dC 44 dA 4 dG.[7] The trans isomer (trans-1), which also
forms adducts with nucleotides in DNA, is released from
50-(2-phosphoryl-1,4-dioxobutane) (DOB), an oxidized abasic
lesion resulting from C50-hydrogen-atom abstraction.[8,11] The
DOB lesion is produced by several antitumour agents and by
g radiation.[11–14] This lesion is a potent irreversible inhibitor
of DNA polymerase b, an enzyme that is a primary component
of base excision repair that is overexpressed in many can-
cers.[15–19] Hence, DOB formation may provide a chemical
basis for the potent cytotoxicity of the antitumor agents that
produce it.
The half-life of DOB in DNA is as short as ,11 h at 378C and
pH 7.2 (10 mM phosphate).[20] Reversible interstrand cross-
linking with dA competes with elimination in duplex DNA,
but the biochemical effects of cross-linking are unknown.[21]
Although cis- and trans-1 form exocyclic adducts with the three
nucleosides containing exocyclic amines, the corresponding
Cis-But-2-en-1,4-dial (cis-1) is a metabolite of the carcino-
gen furan that forms exocyclic adducts with 20-deoxyadenosine
O
O
O
O
O
HO
DOB
OH
[Ox.]
O
trans-1
cis-1
O
DNA
O
H
OH
OH
O
H
O
N
N
O
N
OH
N
N
H
N
N
N
N
H
N
O
H
O
N
H
N
N
H
O
O
O
O
O
4 (dG adduct)
2 (dA adduct)
3 (dC adduct)
O
O
O
Scheme 1.
Ó CSIRO 2011
10.1071/CH10420
0004-9425/11/040438

RESEARCH FRONT
Intramolecular DNA Adduct Formation
439
O
NO2
NVO
O
Oligonucleotide
synthesis and
deprotection
HO
3H
O
NVO
3H
ONV
3H-5
O
OH
3H
ONV
h ν
3H-6
O
O
O
OCH3
OCH3
ONV ꢀ
P
NC
O
3H-DOB
N(iPr)2
Scheme 2.
(a)
S
TBDMSO
HO
S
TBDMSO
O
OH
3H-9
S
S
95%
S
TBDMSO
S
ꢂ
3H-8
7
3H
3H
(1:1)
NVO
O
O
NO2
ONV
3H-5
TBDMSO
O
S
Per Ref. [20]
NC
3H
O
(b)
S
O
48%
P
ONV ꢀ
OCH3
OCH3
3H-7
3H
N(iPr)2
Note: TBDMSO ꢀ t-butyl-dimethyl-silyloxy.
Scheme 3. (a) NaB3H4, MeOH then NaBH4, MeOH; (b) Dess–Martin periodinane.
adduct with dG was unstable to the analytical conditions.[8,22]
Consequently, only dA and dC adducts of trans-but-2-en-1,4-
dial (trans-1) were detected in oxidatively damaged DNA.[8] We
took advantage of our ability to chemically synthesize oligonu-
cleotides containing DOB from a stable precursor (6, Scheme 2)
to enhance detection of but-2-en-1,4-dial (1) adducts of dA, dC,
and dG (2–4) by using tritium labeling.[20]
5ꢁ-d(6NC GTA ATG CAG TCT)
10a N ꢀ G
10b N ꢀ C
10c N ꢀ A
10d N ꢀ T
Fig. 1. Structures 10a–d.
5ꢁ-d(TAA TGG CTA ACG CN44N45 DOB N47 C GTA ATG CAG TCT)
Results and Discussion
3ꢁ-d(ATT ACC GAT TGC GN17N16 N15
N
14 G CAT TAC GTC AGA)
Using DOB as a precursor to cis/trans-but-2-en-1,4-dial (1) is a
more efficient source of the corresponding nucleoside adducts
than furan because the DNA lesion is essentially an intramole-
cular source of the bis-electrophile.[23] Hence, a greater fraction
of 1 reacts with DNA rather than other cellular nucleophiles
(e.g. proteins, thiols). We rationalized that synthesizing a variety
of ternary complexes containing DOB tritiated in a non-
exchangeable position would be useful for probing this possi-
bility by enabling us to examine the distribution of nucleoside
adducts as a function of nucleotide sequence proximal to the
lesion. In addition, using tritiated DOB as a source of cis/trans-
but-2-en-1,4-dial (1) obviates the need for derivatizing the
nucleoside adducts before HPLC analysis. We anticipated that
this would facilitate detection of the dG adducts of 1, as well as
those of dA and dC.
Substrate
N44 N45 DOB N47
N17 N16 N15 N14
11a
11b
11c
11d
A G DOB G
T C
A C DOB C
T G
A A DOB A
T T
T T DOB T
A A
C
C
G
G
T
T
A
A
Fig. 2. Structures 11a–d.
Oligonucleotides (10a–d, Fig. 1, 350 Bq nmolꢀ1) containing
the DOB precursor (3H-6) were synthesized on solid-phase
support using standard synthesis cycles, with two exceptions.
Synthesis of Oligonucleotides Containing Tritiated DOB
The oligonucleotides were prepared as previously described
using the tritiated phosphoramidite (3H-5).[20] Phosphoramidite
3H-5 was synthesized from 7, which was an intermediate in the
synthesis of the unlabeled phosphoramidite (Scheme 3).[20]
Tritium was introduced by reducing the aldehyde with NaB3H4.
This reaction yielded an inseparable 1:1 mixture of the desired
product (3H-8) and that in which the silyl group migrated to the
primary alcohol (3H-9) in 95% yield. Oxidation of the mixture of
3H-8 and 3H-9 with Dess–Martin periodinane yielded the desired
aldehyde (3H-7). The tritiated aldehyde was then carried on to
3H-5 (350 Bq nmolꢀ1) in the manner described by Kodama.[20]
3
The coupling time for H-5 was extended to 5 min and the
detritylation procedure (3% trichloroacetic acid) was not carried
out after this phosphoramidite was coupled at the 50-terminus
of the oligonucleotide. The oligonucleotides were deprotected
using concentrated aqueous ammonia (558C) and purified by
denaturing polyacrylamide gel electrophoresis (PAGE). The
DOB lesion was revealed after hybridizing with the respective
oligonucleotides via photolysis at 350 nm (1 h). The correspond-
ing ternary complexes (11a–d, Figs 1–3) were used immediately
in incubation experiments.

RESEARCH FRONT
440
L. Guan and M. M. Greenberg
Oligonucleotide Elimination Conditions
(,10 nM), which was oxidized by the antitumour antibiotics
calicheamicin and esperamicin (100 mM) or g radiation.[8] In
their study, the yields were determined using a previously
determined amount of DOB in DNA exposed to the same
reaction conditions. They were unable to detect the dG adduct
owing to its instability to the derivatization and analysis condi-
tions.[22] It is important to consider why the adduct yields
observed in our experiment are as much as 75-fold higher.
One difference in our experiments is that we incubated the
DOB-containing DNA before adduct analysis (2 h, 378C, pH
10) in order to induce elimination of 1 and enhance adduct
formation. Peterson, Dedon and coworkers immediately sub-
jected their damaged DNA to the derivatization conditions.
Although their derivatization was carried out for 18 h at 378C
in pH 8.0 buffer, highly nucleophilic O-benzylhydroxylamine
(,5 mM), which would be expected to trap any 1 that is free in
solution and trap any DOB that had not yet undergone elimina-
tion, was present. Hence, the disparity in adduct yields between
the two experiments could be due to our incubating DNA
containing DOB, which produces greater quantities of cis/
trans-but-2-en-1,4-dial (1), before enzyme digestion of the
DNA (Fig. 5).
DOB undergoes b-elimination to form 2-butene-1,4-dial with a
half-life on the order of 11 h under physiologically relevant
conditions.[20] This rate of formation of cis/trans-but-2-en-1,
4-dial (1) is too slow for detecting the nucleoside adducts.
The nucleoside adducts themselves are chemically unstable,
indicating that incubating the ternary complexes for several
half-lives of the elimination reaction would be counter-
productive.[7,24] However, incubating for a shorter time would
yield insufficient quantities of adducts for quantification.
Consequently, cis/trans-but-2-en-1,4-dial (1) elimination from
30-32P-12 was quantified over 2 h between pH 7.2 and 10 at 378C
by denaturing PAGE (Fig. 3). The highest pH in this range was
chosen to carry out subsequent reactions with the tritiated ternary
DOB complexes, because it yielded the most rapid elimination
and adducts 2–4 were stable under these conditions (data not
shown).
Cis/trans-But-2-en-1,4-dial Adduct Formation
Adduct formation was analyzed in four ternary complexes
containing 3H-DOB (12, Fig. 4). The sequence in the vicinity of
DOB was varied to probe for intramolecular effects on reac-
tivity. In two of the ternary complexes, DOB was surrounded by
dG and dC. The other two complexes were rich in dA and dT in
the vicinity of the lesion. After incubating for 2 h as described
above, the DNA was precipitated and digested to nucleosides.
The samples were spiked with diastereomeric mixtures of
unlabeled nucleoside adducts (2–4) that were independently
synthesized as previously described.[7,24] Each nucleoside’s
adducts were separated into two peaks by reverse-phase HPLC.
For quantification purposes, these peaks were collected
separately and the amount of tritium determined by liquid
scintillation counting. The tritium levels were corrected for
background by collecting equal volumes of eluent immediately
before and after the corresponding HPLC peaks and subtracting
the average amounts of radiation in these from the radiation in
the peaks containing the nucleoside adducts. The radioactivity
in the fractions containing adducts were usually two to three-
fold higher than the background fractions.
The amounts of individual cis/trans-but-2-en-1,4-dial
nucleoside adducts (2–4) also did not vary in the manner
Table 1. Total adduct yield as a function of DNA sequence surround-
ing DOB (50-(2-phosphoryl-1,4-dioxobutane))
5ꢁ-d(TAA TGG CTA ACG CN44N45 DOB N47 C GTA ATG CAG TCT)
3ꢁ-d(ATT ACC GAT TGC GN17N16 N15
N
14 G CAT TAC GTC AGA)
Substrate
N
44 N45 DOB N47
Total adduct
yield [%]
N17 N16 N15 N14
11a
11b
11c
11d
A G DOB G
7.5
6.0
5.2
4.6
T C
A C DOB C
T G
A A DOB A
T T
T T DOB T
A A
C
C
G
G
The total adduct yields in the various ternary complexes
ranged between 4.6 and 7.5% in the various ternary complexes
(Table 1). Peterson and coworkers detected ,0.1% of dC (2) and
dA (3) adducts arising from the oxidation of calf thymus DNA
T
T
A
A
Lane
1
2
3
4
Ctl
DOB
Elimination
5
4
3
2
1
0
2 (dA adduct)
3 (dC adduct)
4 (dG adduct)
pH 7.2 8.0 9.0 10.0
% Elimination 12 20 38
7
Fig. 3. DOB (30-32P-12) elimination as a function of pH in phosphate
buffered saline (100 mM, 100 mM NaCl). Each sample was treated with
NaBH4 to stabilize the remaining DOB before gel electrophoresis analysis.
The control (Ctl) was reduced before incubation.
5ꢁ-d(TAA TGG CTA ACG CAA XTC GTA ATG CAG TCT)-32P-3ꢁ
3ꢁ-d(ATT ACG GAT TGC GTT AAG CAT TAG GTC AGA)
11a
11b
11c
11d
Ternary complex
3H-12 X ꢀ DOB
Fig. 5. Yield of adducts 2–4 following incubation of ternary complexes
(11a–d) containing DOB (50-(2-phosphoryl-1,4-dioxobutane)).
Fig. 4. Structure 12.

RESEARCH FRONT
Intramolecular DNA Adduct Formation
441
predicted by reactions of the monomeric nucleosides.[7] The 20-
deoxycytidine adduct (2) did not dominate the product distribu-
tion. The relative amounts were more consistent with those
reported when DNA was treated with various oxidants.[8] When
cis/trans-but-2-en-1,4-dial (1) was released from DOB within
ternary complexes in which the surrounding nucleotides were
rich in dC (11a,b, Fig. 2), adduct 2 was the major product but
significant amounts of dA (3) and dG (4) adducts were also
detected. Furthermore, when the sequence in the vicinity of
DOB was rich in dA, we did not observe consistently high yields
of the corresponding dA adducts (3). Overall, we conclude that
the DNA sequence in the vicinity of DOB has little if any effect
on the distribution of cis/trans-but-2-en-1,4-dial adducts.
However, the exocyclic adduct formation arising from DOB
is clearly more efficient than when the adducts were generated
in bacteria from exogenous but-2-en-1,4-dial.[22] In the experi-
ments described above, the concentration of DOB-containing
DNA (4 mM) represents the maximum possible concentration of
but-2-en-1,4-dial. In contrast, but-2-en-1,4-dial was present at
1 mM in experiments in bacteria.[22] The greater efficiency
for formation of 2–4 in our experiments is attributed in part to
the lack of competition for but-2-en-1,4-dial (1) with cellular
nucleophiles that are present in the bacterial cells. We postulate
formation of 1 from DOB within DNA provides higher yields of
the corresponding nucleoside adducts owing to effective higher
concentration of the electrophile and the absence of other
cellular nucleophiles. The relatively hydrophobic organic mole-
cule may diffuse along the major groove, minor groove, or both
before ultimately being released into solution. However, the rate
constant for reaction of cis/trans-but-2-en-1,4-dial is too small
to compete with diffusion, which is why we do not observe a
strong dependence in adduct distribution as a function of the
DNA sequence surrounding DOB.
Phosphoramidite 3H-5
The tritium-labelled phosphoramidite of the DOB precursor
3
3H-5 was synthesized from compound H-7.[20] The H NMR
1
(CDCl3) and 31P NMR (CDCl3) spectra (OK) of H-5 match
3
previously reported data.[20]
Preparation of 3H-7
MeOH was refluxed with NaBH4 while gently bubbled through
with argon for 24 h at 308C. Sodium was then added and the
solution was refluxed for another 24 h in order to more com-
pletely dry the solvent. MeOH was distilled and stored over
4-A molecular sieves. Compound 7[20] (200 mg, 0.645 mmol) in
˚
distilled MeOH (2 mL) was added to NaB3H4 (8.2 Ci mmolꢀ1
,
100 mCi). NaBH4 (30 mg, 0.8 mmol) was added after stirring the
reaction at 258C for 1 h. The resulting mixture was stirred at the
same temperature for an additional 0.5 h. The reaction mixture
was diluted with diethyl ether and washed with saturated NH4Cl,
followed by brine. The organic phase was dried over Na2SO4
and the solvent was removed under reduced pressure. The
residue was purified by column chromatography (EtOAc/
hexanes, 1:4) to give 8 and 9 (192mg, 0.623 mmol, 95% total) as
a 1:1 mixture. dH (CDCl3) 0.08–0.15 (s, 6H), 0.91–0.92 (s, 12H),
1.78–2.05 and 2.47 (m, 4H), 2.12 (m, 1H), 2.86 (m, 4H), 3.46
(m, 1H), 3.63 (m, 1H), 3.98 and 4.31 (m, 1H), 4.07 (m, 1H).[20]
A mixture of 8 and 9 (192 mg, 0.623 mmol), Dess–Martin
periodinane (396 mg, 0.94 mmol) and anhydrous K2CO3
(138 mg, 1 mmol) in CH2Cl2 (6 mL) was stirred at 258C for
30 min. A mixture of saturated Na2S2O3 and saturated NaHCO3
(1:4) was added to the reaction mixture and stirred vigorously
for 5 min. The organic phase was washed with saturated
NaHCO3, followed by brine, and dried over Na2SO4. The
solvent was removed under reduce pressure and the residue
was purified by column chromatography (EtOAc/hexanes, 1:6)
to give 3H-7 (91.8 mg, 48%, 554 Bq nmolꢀ1). dH (CDCl3) 0.11
(s, 6H), 0.95 (s, 12H), 1.97–2.08 (m, 2H), 2.21 (m, 2H), 2.77–
2.80 (m, 6H), 4.08 (m, 1H), 4.26 (m, 1H), 9.68 (s, 1H).[20]
Conclusions
The DOB lesion is a more efficient source of cis/trans-but-2-en-
1,4-dial nucleoside adducts (2–4) than the carcinogen furan.
This observation is similar to that reported by Dedon et al., who
found that base propenals produced in DNA were a more sig-
nificant source of 20-deoxyguanosine–malondialdehyde adducts
than lipid peroxidation was.[25] Whether this remains true in
cells depends in part on the lifetime of the DOB lesion in this
environment. Efficient DOB repair will reduce its ability to
produce cis/trans-but-2-en-1,4-dial (1), as well as its possible
reaction with the nucleophilic lysine residues within the histone
proteins that make up nucleosomes.[26,27]
Preparation of 1,4-Butenedial Adducts 2–4[7,24]
The 1,4-butenedial adducts were prepared by incubating dC, dA
or dG (5 mM) with 1,4-butenedial (25 mM) in phosphate buffer
(50 mM, pH 7.4) at 378C overnight. The adducts were separated
and collected from reverse-phase HPLC (Delta-Pak C18 150-mm
column, 300 ꢁ 7.8 mm, Waters) (method: the reverse-phase
column was eluted with a linear gradient from 100% solvent A
(H2O) to 75% A and 25% B (acetonitrile) over 30 min with a
flow rate of 2 mL minꢀ1). The adducts were lyophilized to
dryness, resuspended in H2O, and stored at ꢀ808C. The con-
centration of each adduct was determined by measuring the
absorbance at 260 nm and using the extinction coefficient of its
native nucleoside precursor, dC, dA or dG. 2 m/z (electrospray
ionization (ESI)-MS) [Mþ] for C13H17N3O6: calc. 311.29.
Found 311.82. 3: m/z (ESI-MS) [Mþ] for C14H17N5O5: calc.
335.12. Found 335.78. 4: m/z (ESI-M) [Mþ] for C14H17N5O6:
calc. 351.12. Found 389.87 [Mþ þ Mg].
Experimental
Materials and General Methods
Oligonucleotides were prepared on an Applied Biosystems Inc.
394 DNA synthesizer. Commercially available DNA synthesis
reagents were obtained from Glen Research Inc. Snake venom
phosphodiesterases were obtained from USB and Antarctic
phosphatase was obtained from New England Biolabs. NaB3H4
(8.2 Ci mmolꢀ1, 100 mCi) was obtained from Perkin–Elmer.
HPLC was carried out using a Waters system equipped with 515
pumps, and a 2487 multi-wavelength detector controlled by
Empower software. Liquid scintillation counting was carried out
using a Beckman LS6500 counter. Oligonucleotide photolyses
were carried out in a Rayonet photoreactor equipped with 16
lamps with maximum emission at 350 nm.
Effect of pH on DOB Elimination
The ternary complex 30-32P-12 (1 mM) in phosphate-buffered
saline (PBS, 100 mM, 100 mM NaCl, pH 7.2, 8.0, 9.0 or 10.0)
was incubated at 378C for 2 h. Aliquots (5 mL) were removed
and quenched with NaBH4 (2 mL, 500 mM) at 258C for 1 h. The
samples were mixed with formamide loading buffer (15 mL,
90%, 10 mM EDTA). Aliquots (5 mL) of the mixture were

RESEARCH FRONT
442
L. Guan and M. M. Greenberg
loaded onto a 20% denaturing polyacrylamide gel and the pro-
duct was analyzed using a phosphorimager.
[3] M. Tomasz, Y. Palom, Pharmacol. Ther. 1997, 76, 73. doi:10.1016/
S0163-7258(97)00088-0
[4] M. P. Stone, Y.-J. Cho, H. Huang, H.-Y. Kim, I. D. Kozekov,
A. Kozekova, H. Wang, I. G. Minko, R. S. Lloyd, T. M. Harris, C. J.
Rizzo, Acc. Chem. Res. 2008, 41, 793. doi:10.1021/AR700246X
[5] I. D. Kozekov, R. J. Turesky, G. R. Alas, C. M. Harris, T. M. Harris,
C. J. Rizzo, Chem. Res. Toxicol. 2010, 23, 1701. doi:10.1021/
TX100179G
[6] I. G. Minko, I. D. Kozekov, T. M. Harris, C. J. Rizzo, R. S. Lloyd,
M. P. Stone, Chem. Res. Toxicol. 2009, 22, 759. doi:10.1021/
TX9000489
[7] L. Gingipalli, P. C. Dedon, J. Am. Chem. Soc. 2001, 123, 2664.
doi:10.1021/JA0056421
[8] B. Chen, C. C. Vu, M. C. Byrns, P. C. Dedon, L. A. Peterson, Chem.
Res. Toxicol. 2006, 19, 982. doi:10.1021/TX0601197
[9] M. C. Byrns, C. C. Vu, L. A. Peterson, Chem. Res. Toxicol. 2004, 17,
1607. doi:10.1021/TX049866Z
Detection of 1,4-Butenedial Adducts in Ternary Complexes
The ternary complex 11a–d (10 mM, 2 nmol) in PBS buffer
(200 mL, 10 mM, 100 mM NaCl, pH 7.2) was incubated at 378C
overnight. Alternatively, the ternary complex 11a–d (10 mM,
2 nmol) in PBS buffer (200 mL, 100 mM, 100 mM NaCl, pH
10.0) was incubated at 378C for 2 h. DNA was then precipitated
from NaOAc (0.3 M, pH 5.6) and EtOH. The sample was
resuspended inAntarctic phosphatasebuffer(200mL, 50 mMbis-
Tris/propane/HCl, 1 mM MgCl2, 0.1 mM ZnCl2, pH 6.0) and
incubated with 10 units of nuclease P1, 0.28 units of snake venom
phosphodiesterase, and 50 units of Antarctic phosphatase at 378C
for 2 h. The solution was filtered through YM-10 Microcon
filters (Millipore) and washed with water (100mL). The filtrate
was mixed with 2 (100 nM), 3 (20 nM), and 4 (80 nM). Each
adduct was separated and collected from reverse-phase HPLC
(Microsorb-MV C18 5-mm column, 250 ꢁ 4.6 mm). The same
volume of background fractions before and after the adduct peak
was also collected. The HPLC method started at 100% solvent
A (10 mM NH4OAc, pH 6.0) maintained for 5 min with a flow
rate of 1 mL minꢀ1. Then the method changed to a rapid linear
gradient from 100% A to 98% A and 2% B (MeCN) over 1 min,
followed by a linear gradient starting with 98% solvent A, to 80%
A over 60 min. The sample was mixed with liquid scintillation
cocktail (4mL) and equilibrated for 3 h. The radioactivity of
each sample was counted for 2 min using the liquid scintillation
counter.
[10] L. A. Peterson, Drug Metab. Rev. 2006, 38, 615. doi:10.1080/
03602530600959417
[11] B. Chen, T. Bohnert, X. Zhou, P. C. Dedon, Chem. Res. Toxicol. 2004,
17, 1406. doi:10.1021/TX049818E
[12] P. C. Dedon, I. H. Goldberg, Chem. Res. Toxicol. 1992, 5, 311.
doi:10.1021/TX00027A001
[13] H. Kawabata, H. Takeshita, T. Fujiwara, H. Sugiyama, T. Matsuura,
I. Saito, Tetrahedron Lett. 1989, 30, 4263. doi:10.1016/S0040-4039
(01)80706-1
[14] S. Dhar, T. Kodama, M. M. Greenberg, J. Am. Chem. Soc. 2007, 129,
8702. doi:10.1021/JA073014E
[15] D. Starcevic, S. Dalal, J. B. Sweasy, Cell Cycle 2004, 3, 998.
[16] L. Guan, K. Bebenek, T. A. Kunkel, M. M. Greenberg, Biochemistry
2010, 49, 9904. doi:10.1021/BI101533A
[17] L. Guan, M. M. Greenberg, J. Am. Chem. Soc. 2010, 132, 5004.
doi:10.1021/JA101372C
[18] D. M. I. Wilson, D. Barsky, Mutat. Res. 2001, 485, 283.
[19] Y. Matsumoto, K. Kim, Science 1995, 269, 699. doi:10.1126/
SCIENCE.7624801
[20] T. Kodama, M. M. Greenberg, J. Org. Chem. 2005, 70, 9916.
doi:10.1021/JO051666K
[21] L. Guan, M. M. Greenberg, J. Am. Chem. Soc. 2009, 131, 15225.
doi:10.1021/JA9061695
[22] M. C. Byrns, C. C. Vu, J. W. Neidigh, J.-L. Abad, R. A. Jones, L. A.
Peterson, Chem. Res. Toxicol. 2006, 19, 414. doi:10.1021/TX050302K
[23] L.-J. Chen, S. S. Hecht, L. A. Peterson, Chem. Res. Toxicol. 1995, 8,
903. doi:10.1021/TX00049A001
Accessory Publication
NMR spectra of the mixture of 3H-8 and 3H-9, 3H-7, 3H-5 and a
sample HPLC trace of 11a after digestion and spiking with
independently prepared 2–4 are available from the Journal’s
website.
Acknowledgements
This paper is respectfully dedicated to the memory of Professor Athelstan
L. J. Beckwith. We are grateful for financial support from the National
Institute of General Medical Science (GM-063028).
[24] M. C. Byrns, D. P. Predecki, L. A. Peterson, Chem. Res. Toxicol. 2002,
References
15, 373. doi:10.1021/TX0101402
[1] G. M. Blackburn, M. J. Gait, D. Loakes, D. M. Williams, Nucleic
Acids in Chemistry and Biology, 3rd edn 2006 (RSC Publishing:
Cambridge, UK).
[25] X. Zhou, K. Taghizadeh, P. C. Dedon, J. Biol. Chem. 2005, 280, 25377.
doi:10.1074/JBC.M503079200
[26] M. Aso, K. Usui, M. Fukuda, Y. Kakihara, T. Goromaru, H. Suemune,
Org. Lett. 2006, 8, 3183. doi:10.1021/OL060987V
[27] K. Usui, M. Aso, M. Fukuda, H. Suemune, J. Org. Chem. 2008, 73,
241. doi:10.1021/JO702080R
[2] K. S. Gates, in Comprehensive Natural Products Chemistry, DNA and
Aspects of Molecular Biology, Vol. 7 (Ed. E. T. Kool) 1999 (Elsevier:
Amsterdam).