Synthesis of the fully protected phosphoramidite of the benzene-DNA adduct, N2-(4-hydroxyphenyl)-2'-deoxyguanosine and incorporation of the later into DNA oligomers

Article abstract of DOI:10.1080/15257770802258034

N2- (4-Hydroxyphenyl)-2 '-deoxyguanosine-5 '-O-DMT-3 '-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2 '-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2 '-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5 '-O-DMT-3 '-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers. Copyright Taylor & Francis Group, LLC.

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Synthesis of the Fully Protected
Phosphoramidite of the Benzene-DNA
Adduct, N 2-(4-Hydroxyphenyl)-2′-
Deoxyguanosine and Incorporation of the
Later into DNA Oligomers
Ahmed Chenna a , Ramesh C. Gupta b , Radha R. Bonala b , Francis
Johnson b & Bo Hang c
a Monogram Biosciences Inc., South San Francisco, California, USA
b Chem-Master International Inc., Stony Brook, New York, USA
c Department of Genome Stability, Life Sciences Division, Lawrence
Berkeley National Laboratory, University of California, Berkeley,
California, USA
Published online: 11 Aug 2008.
To cite this article: Ahmed Chenna , Ramesh C. Gupta , Radha R. Bonala , Francis Johnson & Bo
Hang (2008) Synthesis of the Fully Protected Phosphoramidite of the Benzene-DNA Adduct, N 2-(4-
Hydroxyphenyl)-2′-Deoxyguanosine and Incorporation of the Later into DNA Oligomers, Nucleosides,
Nucleotides and Nucleic Acids, 27:8, 979-991
To link to this article: http://dx.doi.org/10.1080/15257770802258034
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Nucleosides, Nucleotides and Nucleic Acids, 27:979–991, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802258034
SYNTHESIS OF THE FULLY PROTECTED PHOSPHORAMIDITE
OF THE BENZENE-DNA ADDUCT, N2- (4-HYDROXYPHENYL)-2ꢀ-
DEOXYGUANOSINE AND INCORPORATION OF THE LATER
INTO DNA OLIGOMERS
Ahmed Chenna,1 Ramesh C. Gupta,2 Radha R. Bonala,2 Francis Johnson,2
and Bo Hang3
1Monogram Biosciences Inc., South San Francisco, California, USA
2Chem-Master International Inc., Stony Brook, New York, USA
3Department of Genome Stability, Life Sciences Division, Lawrence Berkeley National
Laboratory, University of California, Berkeley, California, USA
N2-(4-Hydroxyphenyl)-2 ꢁ-deoxyguanosine-5 ꢁ-O-DMT-3 ꢁ-phosphoramidite has been synthesized
2
and used to incorporate the N2-(4-hydroxyphenyl)-2 ꢁ-dG (N2-4-HOPh-dG) into DNA, using
solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium
(Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2 ꢁ-
deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield
and the adduct was converted to the required 5 ꢁ-O-DMT-3 ꢁ-O-phosphoramidite by standard
methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG
adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC.
Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.
Keywords Benzene DNA adduct; hydroquinone; p-benzoquinone; buchwald-hartwig
reaction; N 2-(4-hydroxyphenyl)-2ꢁ-deoxyguanosine
INTRODUCTION
Humans are exposed to benzene from different sources such as auto-
mobile exhaust fumes, cigarette smoke and in industries involved in rubber
manufacture, crude oil, gasoline, and chemical manufacturing. Benzene has
been linked to a number of chronic conditions of the haematopoietic system
Received 12 December 2007; accepted 8 April 2008.
This work was supported by NIH grant CA72079 (to B. H.) and was administrated by the Lawrence
Berkeley National Laboratory under Department of Energy contract DE-AC03-76SF00098.
Address correspondence to Bo Hang, Life Sciences Division, Lawrence Berkeley National Labora-
tory, Berkeley, CA94720. E-mail: Bo hang@lbl.gov
979

980
A. Chenna et al.
of exposed individuals, due to its metabolism in the liver and bone marrow,
to more reactive species.[1] These chronic effects include leucopenia,
thrombocytopenia, pancytopenia, anemia, and myelodysplastic syndrome.
More importantly, benzene is a recognized human leukemogen.[2,3] Several
epidemiologic studies have established a relationship between occupational
exposure to benzene and human leukemia, especially acute myelogenous
leukemia (AML).[4,5]
Inhaled benzene is first metabolized in the liver, where it is converted
by cytochrome P450-2E1 to benzene oxide, then to phenol, hydroquinone
(HQ), and other phenolic metabolites.[6,7] These compounds may travel
to the bone marrow, the main target organ of benzene, where they are
oxidized to genotoxic quinones such as p-benzoquinone (p-BQ) and o-BQ
by the high levels of myeloperoxidase (MPO) in this tissue (Figure 1). The
mutagenic potential of benzene and its major metabolites such as HQ and
p-BQ has been extensively investigated.[8] Studies using mammalian systems
have shown that benzene metabolites induce base substitution and deletion
in various cell lines, animal tissues, and blood from exposed individuals.[8]
The precise mechanisms underlying the benzene-induced mutagenesis
remain poorly understood. There are numerous reports dealing with the
detection of covalent DNA adducts by HQ and p-BQ in vitro.[8,9] These
two compounds are stable metabolites derived from benzene, and p-BQ
is also present in a number of drugs and chemical substances.[10–12] Both
metabolites form exocyclic benzetheno adducts with dG, dA and dC.[13–17]
Although they were not detected in tissues such as the bone marrow by
32P-postlabeling,[18,19] their in vivo formation by similar mechanisms to
those seen in in vitro reactions is expected, given that the yields of these
adducts were high in the latter. Using site-directed mutagenesis, we recently
showed that all three p-BQ adducts are highly mutagenic in Saccharomyces
cerevisiae by predominantly causing deletion mutations.[20] We have also
shown that these p-BQ adducts, when present in DNA, are substrates for
human apurinic/apyrimidinic (AP) endonuclease (APE1), which directly
incises the phosphodiester bond 5ꢁ to the adduct.[21–24]
Although benzene-derived DNA adducts have been identified in vitro,
the in vivo detection of such adducts has been a subject of controversy.
Using 32P-postlabeling, Bauer et al. showed the formation of several DNA
adducts in the livers of rabbits treated with benzene.[25] Later, the Bodell
group discovered in vivo adducts in a human promyelocytic (HL-60) cell
line[18,26] and bone marrow of mice[19] that were treated with benzene,
HQ, or p-BQ. They found that the DNA adducts formed after benzene
administration in mouse bone marrow are identical to those produced in
HL-60 or mouse bone marrow cells treated with HQ or p-BQ. Moreover,
the observed induction of toxicity in the bone marrow was paralleled by
formation of DNA adducts.[19] By 32P-postlabeling, the principal adduct
found in the cells targeted by benzene has the same chromatographic

Synthesis and Insertion of N2-4-HOPh-dG into DNA
981
FIGURE 1 The metabolic route from benzene to the formation of HQ and p-BQ. These two
stable metabolites may react with guanine in the bone marrow to form N 2-(4-hydroxyphenyl)-2ꢁ-
deoxyguanosine (N 2-4-HOPh-dG).

982
A. Chenna et al.
properties as N 2-(4-hydroxyphenyl)-2ꢁ-deoxyguanosine-3ꢁ-phosphate.[18,26]
This adduct can also be formed in vitro by allowing guanosine 3ꢁ-phosphate
to react with p-BQ.[18,26] To date, the biochemical properties and biological
importance of this adduct await investigation.
In order to understand the molecular mechanism by which HQ and
p-BQ exert their mutagenic effects, it is important to study their princi-
pal adduct formed in vivo, N 2-(4-hydroxyphenyl)-2ꢁ-deoxyguanosine (N 2-
4-HOPh-dG), regarding its potential effects on DNA replication and repair.
Such studies would be greatly facilitated with oligonucleotides containing a
site-specific adduct. Previously, we have synthesized the phosphoramidites
of exocyclic p-BQ-dA, p-BQ-dC and one of the p-BQ-dG adducts and
incorporated them site-specifically into oligomeric DNA.[27,28] In this work,
we report for the first time the synthesis of N 2-4-HOPh-dG-phosphoramidite
and its incorporation into defined DNA oligonucleotides.
RESULTS AND DISCUSSION
Previously, Pongracz and Bodell[26] reported the synthesis of N 2-(4-
hydroxyphenyl)-2ꢁ-deoxyguanosine-3ꢁ-phosphate as the 3ꢁ-phosphate. We
have now successfully synthesized the fully-protected N 2-(4-hydroxyphenyl)-
2ꢁ-deoxyguanosine-3ꢁ-phosohoramidite 7, by a completely new method
(Scheme 1), which gave better yields. Our approach is similar to that used
previously for the synthesis and incorporation of the 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP) adducts into DNA in which the key
procedure is a Buchwald-Hartwig (BH) reaction.[29,30] Compound 1 when
coupled with 4-isobutyryloxybromobenzene 2 under the conditions of the
Buchwald-Hartwig reaction gave compound 3 in 80% yield. Removal of
the 6-O-benzyl group from the purine moiety was then accomplished by
hydrogenolysis over a palladium catalyst to give compound 4, which, after
purification by column chromatography, was obtained in 88% yield. The
silyl protecting groups of compound 4 were removed by HF in pyridine,
to give compound 5 in 76% yield after purification. The electrospray mass
spectrum of this compound showed an intense protonated molecular ion
at m/z 430 (M+H)+ and the natriated species at m/z at 452 (M + Na)+
(Figure 2). Reaction of compound 5 with DMT-Cl in pyridine produced
compound 6 in 90% yield, which was then allowed to react with (2-
cyanoethoxy)-bis(N ,N -diisopropylamino)phosphane to produce the fully
protected phosphoramidite compound 7. All of the above compounds were
1
identified by MS, H NMR, 13C NMR and in the case of compound 7, 31P
NMR analysis was used as well. (See 1H NMR of 7 in Figure 3.) The latter was
then used to introduce the xenonucleoside into defined oligonucleotides
by DNA synthesizer. The composition of the oligomers was confirmed
after enzymatic digestion of the DNA oligomers to nucleosides, followed

Synthesis and Insertion of N2-4-HOPh-dG into DNA
983
SCHEME 1 The synthesis of the fully protected N 2-(4-hydroxyphenyl)-2ꢁ-deoxyguanosine-3ꢁ-
phosphoramidite 7.
by HPLC analysis, which showed that the N 2-4-HOPh-dG residual survived
the conditions used in the DNA synthesis and the deprotection procedure
(Figure 4). Based on the calculation of the integrated area of each HPLC
peak of the nucleosides and the modified base (Figure 4) and by using
the extinction coefficient for each nucleoside, we found that the 40-mer
contains 9.9 dC, 9.4 dG, 12.4 T, 7.4 dA, and 0.9 N 2-4-HOPh-dG. The actual
composition of the 40-mer is 10 dC, 9 dG, 12 T, 8 dA, and 1 N 2-4-HOPh-dG.

984
A. Chenna et al.
FIGURE 2 Electrospray mass spectrum of N 2-4-HOPh-dG (5).
The biochemical work is underway to evaluate the mutagenic potential of
this adduct as well as possible repair mechanisms that acts on it using these
oligonucleotides.
EXPERIMENTAL
(Caution: The organic solvents used in this work should be stored and
used in a well-ventilated hood.)
Chemicals and Reagents
All reagents and solvents employed were of commercial grade and
were used as such, unless otherwise specified. NMR (1H and 13C) spectra
were recorded on a Gemini 300 spectrometer. Samples prepared for NMR
analysis were dissolved in CDCl3 or DMSO-d6. Chemical shifts are reported
in ppm relative to TMS in the proton spectra and to the deuterated
solvent in the carbon spectra. Mass spectra were recorded on a Micromass
Trio in fast atom bombardment (FAB) mode. Thin-layer chromatography

Synthesis and Insertion of N2-4-HOPh-dG into DNA
985
FIGURE 3 1H NMR of N 2-(4-hydroxyphenyl)-2ꢁ-deoxyguanosine-3ꢁ-phosohoramidite (7).
(TLC) was performed on silica gel sheets (riedel-deHae¨n, Sleeze, Germany)
containing a fluorescent indicator. Components were visualized by UV
light (λ = 254 nm) or by spraying with a solution of phosphomolybdic
˚
acid. Flash column chromatography separations were carried out on 60 A
(230–400 mesh) silica gel (Silica-P Flash Silica Gel obtained from Silicycle,
Quebec, QC, Canada). All experiments dealing with moisture or air-sensitive
compounds were conducted under dry nitrogen. The starting materials
and reagents, unless otherwise specified, were the best commercial grades
available (Aldrich, Fluka) and were used without further purification. After
purification all new products showed a single spot on TLC analysis.
HPLC
Solvent systems included acetonitrile (solvent A), triethylammonium
acetate (0.1 M, pH 7.0, solvent B), and potassium phosphate buffer (0.01
M, pH 4.5, solvent C). System 1: Oligonucleotides were purified using a
PRP-1-C18 column (305 × 7 mm, 10 µm, Hamilton Corp.). The initial
concentration of solvent A was 10% and solvent B was 90%, then solvent A
was increased linearly to 38% over 40 minutes at a flow rate of 3 ml/minute.
System 2: This was used for the analysis of the enzyme digest of the
oligonucleotides and was performed with a Supelcosil LC-18-DB column

986
A. Chenna et al.
FIGURE 4 A) HPLC profile of 2ꢁ-deoxynucleosides obtained as a result of enzymatic digestion of the
N 2-4-HOPh-dG-containing 40-mer. B) HPLC profile of N 2-4-HOPh-dG.
(25 × 0.46 cm, 5 µm, Supelco Inc.) using 0% solvent A, 100% solvent C
at start. Solvent A was linearly increased to 12% over 30 minutes, then to
40% over 15 minutes where it was held for 10 minutes at a flow rate of 1
ml/minute. The HPLC instrument was an Agilent 1100 series.
Ultraviolet spectra were recorded on an Agilent 8453 spectrophotome-
ter using 0.2 cm cuvette. TLC was performed on silica gel 60, F254 plates.

Synthesis and Insertion of N2-4-HOPh-dG into DNA
987
Column chromatography was performed using silica gel 60 with elution
under pressure.
Chemical Syntheses (Scheme 1)
N2-(4-Isobutyryloxyphenyl)-O6-benzyl-3ꢁ,5ꢁ-bis-O-(tert-butyldimethylsilyl)-2ꢁ-
deoxyguanosine (3)
An oven-dried 100 mL two-necked round-bottomed flask was charged
with the protected purine nucleoside 1[31] (1.17 g. 2 mmol), cesium
carbonate (0.92 g, 2.8 mmol), Pd2(dba)3 (230 mg, 0.25 mmol), xantphos
(480 mg, 0.8 mmol), 4-isobutyryloxybromobenzene (2, 0.724 g, 2.98 mmol)
and toluene (15 mL) under a nitrogen atmosphere. This mixture was stirred
for 30 minutes at room temperature, later heated at 80◦C for 6 hours. It
was cooled to room temperature, the solids were filtered, and the solid
cake was washed with ethyl acetate. The filtrate was evaporated to dryness
and the residue was purified by silica gel column chromatography using
1
CH2Cl2:ethyl acetate (98:2) as the eluant to give pure 3 (1.2 g, 80%). H
NMR (CDCl3): δ 8.09 (s, 1H), 7.67 (d, 2H), 7.55 (m, 2H),7.45–7.33 (m,
3H), 7.28 (br s, 1H), 7.09 (d, 2H), 6.46 (t, 1H), 5.68 (s, 2H), 4.64 (m, 1H),
4.08 (m, 1H), 3.88–3.85 (m, 2H), 2.87 (m, 1H), 2.72–2.39 (m, 2H), 1.40
(s, 3H), 1.38 (s, 3H), 0.98 (s, 9H), 0.97 (s, 9H), 0.16 (s, 6H), 0.15 (s, 6H).
13C NMR (CDCl3): δ 175.43, 160.15, 155.26, 152.81, 145.11, 137.79, 137.35,
136.08, 128.03, 127.68, 127.60, 121.24, 119.37, 116.09, 87.35, 83.61, 77.42,
77.00, 76.57, 71.64, 67.79, 62.53, 41.01, 33.80, 25.68, 25.47, 18.67, 18.09,
17.67, −4.94, −5.06, −5.66, −5.77. ESI (M + H) 748.
N2-(4-Isobutyryloxyphenyl)-3ꢁ,5ꢁ-bis-O-(tert-butyldimethylsilyl)-2ꢁ-
deoxyguanosine (4)
To a solution of 3 (1.2 g, 1.6 mmol) in ethyl acetate (50 mL) was added
a 10% palladium-on-carbon (150 mg) catalyst. The flask was evacuated (50
Torr) and flushed with hydrogen three times. The mixture was shaken
under hydrogen for 16 hours at 50 psi, filtered through a pad of Celite to
remove the catalyst, and then concentrated under reduced pressure. The
residue was purified by column chromatography using 3% methanol in
methylene chloride to obtain the pure debenzylated product 4 as a glassy
solid (0.930 gm 88%). 1H NMR (CDCl3): δ 11.76 (br s, 1H), 9.75 (br s, 1H),
8. 14 (d, 2H), 8.09 (s, 1H), 7.10 (d, 2H), 6.41 (t, 1H), 4.63 (m, 1H), 4.07
(m, 2H), 2.87 (m, 1H), 2.52 (m, 2H), 1.40 (s, 3H), 1.38 (s, 3H), 1.00 (s, 9H),
0.98 (s, 9H), 0.18 (s, 6H), 0.16 (s, 1H). 13C NMR (CDCl3): δ 175.54, 158.83,
150.62, 149.57, 146.05, 136.46, 136.31, 121.33, 121.02, 118.16, 87.64, 84.04,
71.76, 62.75, 41.71, 34.09, 25.92, 25.71, 18.94, 18.35, 17.90, −4.68, −4.82,
−5.41, −5.53. ESI (M + H) 658.
Preparation of N2-(4-isobutyryloxyphenyl)-2ꢁ-deoxyguanosine (5). To an ice-
cold solution of 4 (0.9 g, 1.37 mmol) in pyridine (20 mL) was added HF/Py

988
A. Chenna et al.
(2.2 mL, 70% of HF in Py from Aldrich Chemical Co.) over a period of
3 minutes. The mixture was stirred at room temperature overnight, then
poured into ice-cold water (100 mL) containing NaHCO3 (6.5 g) and
stirred for 2 hours. The reaction mixture was evaporated to dryness and
co-evaporated with methanol three times and the residual material was
adsorbed onto silica gel using methanol as a solvent. It was purified by silica
gel using 15% methanol in methylene chloride to obtain the pure 5 as a
white solid (450 mg, 76%). 1H NMR (DMSO-d6): δ 10.67 (br s, 1H), 9.01 (s,
1H), 8.03 (s, 1H), 7.61 (d, 2H), 7.09 (d, 2H), 6.195 (t, 1H), 5.27 (br s, 1H),
4.89 (br s, 1H), 3.81 (m, 1H), 3.50 (m, 2H), 2.78 (m, 1H), 2.59 (m, 1H),
2.28 (m, 1H), 1.22 (s, 3H), 1.20 (s, 3H). 13C NMR (DMSO-d6): δ 175.96,
157.14, 150.09, 149.97, 146.32, 137.18, 122.73, 120.95, 118.97, 88.35, 83.64,
71.31, 62.29, 33.97, 19.38. ESI (M+H) 429.
N2-(4-Isobutyryloxyphenyl)-5ꢁ-O-(4.4ꢁ-dimethoxytrityl)-2ꢁ-deoxyguanosine (6)
To a solution of 5 (0.428 g, 1 mmol) in pyridine (15 mL) was added
DMT chloride (0.440 g, 1.3 mmol) as a solid and the mixture was stirred
at room temperature for 3 hours. TLC (CH2Cl2: MeOH: 85:15) of the
mixture showed the reaction to be ∼90% complete. The reaction was
quenched by adding methanol (5 mL) and the mixture was stirred for
30 minutes at room temperature, and then concentrated under reduced
pressure. To the residue was added aqueous NaHCO3 (25 mL) and the
mixture was extracted with methylene chloride. The methylene chloride
extract was dried over anhydrous MgSO4, filtered, and concentrated. The
crude product was purified by column chromatography using TEA-treated
silica gel and CH2Cl2: MeOH (95:5) as the eluant to give pure 6 (550 mg,
90% yield based on consumed starting material). The column was further
eluted with 15% methanol in methylene chloride to recover the unreacted
starting material (70 mg). 1H NMR (CDCl3): δ 10.01 (br s, 1H), 7.75 (s, 1H),
7.58 (d, 2H), 7.41–7.16 (m, 10H), 6.89 (d, 2H), 6.79 (d, 4H), 6.15 (t, 1H),
4.46 (m, 1H), 4.05 (m, 1H), 3.73 (s, 6H), 3.27 (m, 2H), 2.84 (m, 1H), 2.72
(m, 1H), 2.41 (m, 1H). 13C NMR (CDCl3): δ 176.51, 158.38, 150.19, 149.95,
146.30, 144.68, 136.73, 135.90, 135.86, 129.95, 128.13, 127.69, 126.71,
123.31, 121.49, 118.59, 113.00, 86.23, 85.61, 83.98, 71.44, 64.25, 55.09, 39.52,
34.07, 18.83. ESI (M+H) 732.
N2-(4-Isobutyryloxyphenyl)-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-O-[N,N-
diisopropylamino-(2-cyanoethoxy)-phosphinyl]-2ꢁ-deoxyguanosine (7)
Compound 6 (256 mg, 0.35 mmol) was co-evaporated with dry toluene
(3 × 10 mL), and the residue was redissolved in dry methylene chlo-
ride (10 mL). Tetrazole (28 mg, 0.40 mmole) was then added followed
by (2-cyanoethoxy)- bis(N ,N -diisopropylamino)phosphane (147 mg, 0.49
mmole). The reaction mixture was stirred at room temperature for 2 hours
under nitrogen and then poured into aqueous NaHCO3 (10%, 10 mL). The

Synthesis and Insertion of N2-4-HOPh-dG into DNA
989
methylene chloride layer was separated and the aqueous layer was extracted
once with methylene chloride (20 mL). The combined methylene chloride
extract was dried over Na2SO4, filtered and concentrated under reduced
pressure to obtain the desired crude product 7 as an oil. The crude residue
was purified on a silica gel column pretreated with triethylamine, using
methylene chloride:acetone (3:2) as the eluant. The fractions containing
the pure material were collected and concentrated under reduced pressure
and the resulting solid was triturated with isorpropyl ether and filtered to
give pure 7 as a white solid (240 mg, 73%). 1H NMR (CDCl3): δ 8.76 (br s,
1H), 8. 02 (br s, 1H), 8.01 (d, 1H), 7.88 (d, 1H), 7.48–7.25 (m, 10H), 7.02
(d, 2H), 6.85 (m. 4H), 6.41 (t, 1H), 4.71 (m, 1H), 4.34 (m, 1H), 3.80 (s,
6H), 3.65–3.74 (m, 5H), 3.41 (m, 2H), 3.09 (m, 1H), 2.87–2.47 (m, 3H),
1.38–1.16 (m, 18H). 13C NMR (CDCl3): δ 175.62, 158.94, 158.55, 150.92,
149.72, 146.26, 144.52, 136.22, 135.63, 135.53, 130.13, 130.07, 130.02,
128.16, 128.10, 127.85, 126.90, 121.54, 121.26, 118.46, 117.61, 113.16, 86.48,
85.81, 84.15, 74.01, 63.66, 58.40, 58.21, 55.17, 46.13, 43.39, 43.34, 43.27,
43.21, 40.24, 34.12, 24.58, 24.55, 24.51, 20.35, 20.27, 20.18, 18.96. 31P NMR
(CDCl3): δ 150.09, 149.60.
Solid-Phase Synthesis of DNA Oligonucleotides
The fully-protected phosphoramidite compound 7 was used to synthe-
size three different oligonucleotides at the 1 µmole scale: a 22-mer, a
25-mer, and a 40-mer using an Applied Biosystem 394 automated DNA
synthesizer by standard phosphoramidite chemistry. Phenoxyacetyl (PAC)
was used as the N -ptotecting group in the synthesis of the DNA oligomers.
Because amidite 7 has very limited solubility in acetonitrile, dry methylene
chloride (100%) was used to dissolve the amidite 7, which was used in
the DNA synthesis. The coupling time for the amidite 7 was increased to
15 minutes, and the coupling efficiency at the N 2-4-HOPh-dG step was
97% for all oligonucleotides. The overall yields of the oligonucleotides
were 86% for the 22-mer, 80% for the 25-mer, and 65% for the 40-mer.
The oligonucleotides were then cleaved from the resin and deprotected by
means of 28% ammonium hydroxide containing 1% of 2-mercaptoethanol
for 2 hours at 65◦C. The latter reagent prevents oxidative degradation of the
N 2-4-HOPh-dG lesion. The oligonucleotides were purified by reverse phase
HPLC on a C-18 column. The sequences of the DNA oligomers synthesized
are as follows:
1: 5ꢁ-GTAAGCTXGATCCTCTAGAGCG-3ꢁ (22-mer)
2: 5ꢁ-CCGCTAXCGGGTACCGAGCTCGAAT-3ꢁ (25-mer)
3: 5ꢁ-TTGCTTTGTCACCCAGGCTXGACTGCAGTGGTACAATCAT-3ꢁ (40-
mer)
X = N 2-4-HOPh-dG

990
A. Chenna et al.
Enzymatic Digestion of the Oligomers
The composition of the oligomers was confirmed by enzymatic digestion
of the DNA oligomers to nucleosides, which were identified by HPLC
analysis. A defined oligonucleotide (1.0 A260 unit) was dissolved in 44 µL of
deionized water, 0.8 µL of 1 M MgCl2, and 3.5 µL of 0.5 M Tris-HCl buffer
(pH 7.5) and digested with snake venom phosphodiesterase (2.5 µL) and
bacterial alkaline phosphatase (4.0 µL) at 37 ◦C for 16 hours. The digested
mixture was then analyzed on a C-18 reverse-phase HPLC Supelcosil column
using solvent system 2 noted above.
REFERENCES
1. NTP Toxicology and Carcinogenesis Studies of Benzene (CAS No. 71-43-2) in F344/N Rats and
B6C3F1 Mice (Gavage Studies). Natl. Toxicol. Program Tech. Rep. Ser. 1986, 289, 1–277.
2. IARC. IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans. Some industrial
chemicals and dyestuffs. International Agency for Research on Cancer: Lyon, France, 1982, vol. 29.
3. NTP. 11th Report on carcinogens. U.S. Department of Health and Human Services, National Toxicology
Program, Atlanta, GA, 2005.
4. Yin, S.-N.; Hayes, R.; Linet, M.; Li, G.; Dosemeci, M.; Travis, L.; Zhang, Z.; Li, D.; Chow, W.;
Wacholder, S.; Blot, W. Recent studies of human exposure to benzene in China and Europe An
expanded cohort study of cancer among benzene-exposed workers in China. Environ. Health Perspect.
1996, 1339–1341.
5. Glass, D.C.; Gray, C.N.; Jolley, D.J.; Gibbons, C.; Sim, M.R.; Fritschi, L.; Adams, G.G.; Bisby, J.A.;
Manuell, R. Leukemia risk associated with low-level benzene exposure. Epidemiology 2003, 14,
569–577.
6. Snyder, R.; Hedli, C.C. An overview of benzene metabolism. Environ. Health Perspect. 1996, 104
(Suppl. 6), 1165–1171.
7. Lovern, M.R.; Cole, C.E.; Schlosser, P.M. A review of quantitative studies of benzene metabolism.
Crit. Rev. Toxicol. 2001, 31, 285–311.
8. Whysner, J.; Reddy, M.V.; Ross, P.M.; Mohan, M.; Lax, E.A. Genotoxicity of benzene and its
metabolites. Mutat. Res. 2004, 566, 99–130.
9. Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. Proceedings of the 2nd international
conference Heidelberg, Germany, September 1998. IARC Sci Publ 1999, 150, 1–361.
10. Zheng, J.; Hanzlik, R.P. Dihydroxylated mercapturic acid metabolites of bromobenzene. Chem. Res.
Toxicol. 1992, 5, 561–567.
11. Pascoe, G.A.; Calleman, C.J.; Baille, T.A. Identification of S-(2,5-dihydroxyphenyl)-cysteine and
S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine as urinary metabolites of acetaminophen in the mouse.
Evidence for p-benzoquinone as a reactive intermediate in acetaminophen metabolism. Chem. Biol.
Interact. 1988, 68, 85–98.
12. McDonald, T.A.; Waidyanatha, S.; Rappaport, S.M. Measurement of adducts of benzoquinone with
hemoglobin and albumin. Carcinogenesis 1993, 14, 1927–1932.
13. Jowa, L.; Winkle, S.; Kalf, G.; Witz, G.; Snyder, R. Deoxyguanosine adducts formed from benzo-
quinone and hydroquinone. Adv. Exp. Med. Biol. 1986, 197, 825–832.
14. Jowa, L.; Witz, G.; Snyder, R.; Winkle, S.; Kalf, G.F. Synthesis and characterization of deoxyguanosine-
benzoquinone adducts. J. Appl. Toxicol. 1990, 10, 47–54.
15. Pongracz, K.; Bodell, W.J. Detection of 3ꢁ-hydroxy-1,N6-benzetheno-2ꢁ-deoxyadenosine 3ꢁ-phosphate
by 32P postlabeling of DNA reacted with p-benzoquinone. Chem. Res. Toxicol. 1991, 4, 199–
202.
16. Pongracz, K.; Kaur, S.; Burlingame, A.L.; Bodell, W.J. Detection of (3ꢁ-hydroxy)-3,N4-benzetheno-2ꢁ-
deoxycytidine-3ꢁ-phosphate by 32P-postlabeling of DNA reacted with p-benzoquinone. Carcinogenesis
1990, 11, 1469–1472.
17. Gaskell, M.; Jukes, R.; Jones, D.J.; Martin, E.A.; Farmer, P.B. Identification and characterization
of (3ꢁꢁ,4ꢁꢁ-dihydroxy)-1,N2-benzetheno-2ꢁ-deoxyguanosine 3¢-monophosphate, a novel DNA adduct

Synthesis and Insertion of N2-4-HOPh-dG into DNA
991
formed by benzene metabolites. Chem. Res. Toxicol. 2002, 15, 1088–1095.
18. Levay, G.; Pongracz, K.; Bodell, W.J. Detection of DNA adducts in HL-60 cells treated with
hydroquinone and p-benzoquinone by 32P-postlabeling. Carcinogenesis 1991, 12, 1181–1186.
19. Bodell, W.J.; Pathak, D.N.; Levay, G.; Ye, Q.; Pongracz, K. Investigation of the DNA adducts formed
in B6C3F1 mice treated with benzene: implications for molecular dosimetry. Environ. Health Perspect.
1996, 104 (Suppl. 6), 1189–1193.
20. Xie, Z.; Zhang, Y.; Guliaev, A.B.; Shen, H.; Hang, B.; Singer, B.; Wang, Z. The p-benzoquinone DNA
adducts derived from benzene are highly mutagenic. DNA Repair (Amst) 2005, 4, 1399–1409.
21. Chenna, A.; Hang, B.; Rydberg, B.; Kim, E.; Pongracz, K.; Bodell, W.J.; Singer, B. The benzene
metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from
human cells that differs from an ethenoadenine glycosylase. Proc. Natl. Acad. Sci. USA 1995, 92,
5890–5894.
22. Hang, B.; Chenna, A.; Fraenkel-Conrat, H.; Singer, B. An unusual mechanism for the major
human apurinic/apyrimidinic (AP) endonuclease involving 5¢ cleavage of DNA containing a
benzene-derived exocyclic adduct in the absence of an AP site. Proc. Natl. Acad. Sci. USA 1996, 93,
13737–13741.
23. Hang, B.; Chenna, A.; Sagi, J.; Singer, B. Differential cleavage of oligonucleotides containing the
benzene-derived adduct, 1,N6-benzetheno-dA, by the major human AP endonuclease HAP1 and
Escherichia coli exonuclease III and endonuclease IV. Carcinogenesis 1998, 19, 1339–1343.
24. Guliaev, A.B.; Hang, B.; Singer, B. Structural insights by molecular dynamics simulations into
specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C.
Nucleic. Acids. Res. 2004, 32, 2844–2852.
25. Bauer, H.; Dimitriadis, E.A.; Snyder, R. An in vivo study of benzene metabolite DNA adduct
formation in liver of male New Zealand rabbits. Arch. Toxicol. 1989, 63, 209–213.
26. Pongracz, K.; Bodell, W.J. Synthesis of N2-(4-hydroxyphenyl)-2ꢁ-deoxyguanosine 3¢-phosphate: com-
parison by 32P-postlabeling with the DNA adduct formed in HL-60 cells treated with hydroquinone.
Chem. Res. Toxicol. 1996, 9, 593–598.
27. Chenna, A.; Singer, B. Large scale synthesis of p-benzoquinone-2ꢁ-deoxycytidine and p-
benzoquinone-2ꢁ-deoxyadenosine adducts and their site-specific incorporation into DNA oligonu-
cleotides. Chem. Res. Toxicol. 1995, 8, 865–874.
28. Chenna, A.; Singer, B. Synthesis of a benzene metabolite adduct, 3ꢁꢁ-hydroxy-1,N2-benzetheno-2ꢁ-
deoxyguanosine, and its site-specific incorporation into DNA oligonucleotides. Chem. Res. Toxicol.
1997, 10, 165–171.
29. Lakshman, M.K. Synthesis of biologically important nucleoside analogs by Palladium-catalyzed C-N
bond-formation. Curr. Org. Synth. 2005, 2, 83–112.
30. Bonala, R.; Torres, M.C.; Iden, C.R.; Johnson, F. Synthesis of the PhIP adduct of 2ꢁ-deoxyguanosine
and its incorporation into oligomeric DNA. Chem. Res. Toxicol. 2006, 19, 734–738.
31. Harwood, E.A.; Sigurdsson, S.T.; Edfeldt, N.B.F.; Reid, B.R.; Hopkins, P.B. Synthesis and preliminary
structural characterization of nitrous acid interstrand cross-linked duplex DNA. J. Am. Chem. Soc.
1999, 121, 5081–5082.