6-Nitrochrysene-Derived C8-2′-Deoxyadenosine Adduct: Synthesis of Site-Specific Oligodeoxynucleotides and Mutagenicity in Escherichia coli

Article abstract of DOI:10.1021/acs.chemrestox.9b00429

6-Nitrochrysene (6-NC), the most potent carcinogen evaluated by the newborn mouse assay, is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields three major DNA adducts: at the C8 and N² positions of 2′-deoxyguanosine (dG), N-(dG-8-yl)-6-AC and 5-(dG-N²-yl)-6-AC, and at the C8 position of 2′-deoxyadenosine (dA), N-(dA-8-yl)-6-AC. A nucleotide excision repair assay demonstrated that N-(dA-8-yl)-6-AC is repaired much more slowly than many other bulky DNA adducts, including the other DNA adducts formed by 6-NC. But neither the total synthesis nor evaluation of other biological activities of this dA adduct has ever been reported. Herein, we report a convenient synthesis of the 6-NC-derived dA adduct by employing the Buchwald-Hartwig coupling strategy, which provided a high yield of the protected N-(dA-8-yl)-6-AC. The deprotected nucleoside showed syn conformational preference by NMR spectroscopy. Following DMT protection of the 5′-hydroxyl, N-(dA-8-yl)-6-AC was converted to its 3′-phosphoramidite, which was used to prepare oligonucleotides containing a single N-(dA-8-yl)-6-AC adduct. Circular dichroism spectra of the adducted duplex showed only a slight departure from the B-DNA helix profile of the control duplex. The 15-mer N-(dA-8-yl)-6-AC oligonucleotide was used to construct a single-stranded plasmid vector containing a single adduct, which was replicated in Escherichia coli. Viability of the adducted construct was ～60% of the control, indicating slower translesion synthesis of the adduct, which increased to nearly 90% upon induction of the SOS functions. Without SOS, the mutation frequency (MF) of the adduct was 5.2%, including 2.9% targeted and 2.3% semi-targeted mutations. With SOS, the targeted MF increased 3-fold to 9.0%, whereas semi-targeted mutation increased only marginally to 3.2%. The major type of targeted mutation was A*→G in both uninduced and SOS-induced cells.

Full text of DOI:10.1021/acs.chemrestox.9b00429

pubs.acs.org/crt
Article
6‑Nitrochrysene-Derived C8-2′-Deoxyadenosine Adduct: Synthesis
of Site-Specific Oligodeoxynucleotides and Mutagenicity in
Escherichia coli
Brent V. Powell and Ashis K. Basu*
Cite This: https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: 6-Nitrochrysene (6-NC), the most potent carcinogen evaluated
by the newborn mouse assay, is metabolically activated by nitroreduction and a
combination of ring oxidation and nitroreduction pathways. The nitroreduction
pathway yields three major DNA adducts: at the C8 and N2 positions of 2′-
deoxyguanosine (dG), N-(dG-8-yl)-6-AC and 5-(dG-N2-yl)-6-AC, and at the C8
position of 2′-deoxyadenosine (dA), N-(dA-8-yl)-6-AC. A nucleotide excision
repair assay demonstrated that N-(dA-8-yl)-6-AC is repaired much more slowly
than many other bulky DNA adducts, including the other DNA adducts formed
by 6-NC. But neither the total synthesis nor evaluation of other biological
activities of this dA adduct has ever been reported. Herein, we report a
convenient synthesis of the 6-NC-derived dA adduct by employing the
Buchwald−Hartwig coupling strategy, which provided a high yield of the
protected N-(dA-8-yl)-6-AC. The deprotected nucleoside showed syn conforma-
tional preference by NMR spectroscopy. Following DMT protection of the 5′-hydroxyl, N-(dA-8-yl)-6-AC was converted to its 3′-
phosphoramidite, which was used to prepare oligonucleotides containing a single N-(dA-8-yl)-6-AC adduct. Circular dichroism
spectra of the adducted duplex showed only a slight departure from the B-DNA helix profile of the control duplex. The 15-mer N-
(dA-8-yl)-6-AC oligonucleotide was used to construct a single-stranded plasmid vector containing a single adduct, which was
replicated in Escherichia coli. Viability of the adducted construct was ∼60% of the control, indicating slower translesion synthesis of
the adduct, which increased to nearly 90% upon induction of the SOS functions. Without SOS, the mutation frequency (MF) of the
adduct was 5.2%, including 2.9% targeted and 2.3% semi-targeted mutations. With SOS, the targeted MF increased 3-fold to 9.0%,
whereas semi-targeted mutation increased only marginally to 3.2%. The major type of targeted mutation was A*→G in both
uninduced and SOS-induced cells.
ion reacts with the nucleophilic sites of purine bases to
generate DNA adducts, N-(dG-8-yl)-6-AC (1), 5-(dG-N2-yl)-
6-AC (2), and N-(dA-8-yl)-6-AC (3).13 N-(dA-8-yl)-6-AC (3)
is believed to give rise to the corresponding 2′-deoxyinosine
adduct, but whether the process of deamination is enzymatic
or non-enzymatic has never been established.13
Additional adducts are formed by a combination of ring
oxidation and nitroreduction.16,17 DNA lesions derived from 6-
NC can play important roles in the development of human
cancer if they are not repaired via cellular repair pathways prior
to DNA replication.18,19 These lesions can induce mutations in
crucial sequences of cancer genes. Mutations in an oncogene, a
tumor-suppressor gene such as p53, or a gene that controls the
cell cycle can lead to uncontrolled cell growth, resulting in
INTRODUCTION
■
Nitropolycyclic aromatic hydrocarbons (NO2-PAHs) have
been detected in a variety of environmental samples, including
diesel exhaust.1−3 Many of these compounds are potent
mutagens and carcinogens in laboratory animals.4−6 6-
Nitrochrysene (6-NC) is less abundant than other NO2-
PAHs in the environment. However, it is the most potent
NO2-PAH compound ever tested in newborn mouse assay.7 6-
NC is a potent mutagen in bacteria.8 It is an exceptionally
potent carcinogen in newborn mice, and it induces mammary
carcinoma in rats.9,10 The genotoxicity of 6-NC is derived from
its ability to chemically damage 2′-deoxyguanosine (dG) and
2′-deoxyadenosine (dA) in DNA to generate carcinogen−
DNA adducts.11−13 Several in vivo studies in mice and rats
have demonstrated that 6-NC can be activated by two major
pathways:14−16 a nitroreduction and a nitroreduction−ring
oxidation tethered pathway. The nitroreduction pathway
involves a simple nitroreduction of 6-NC to form the
corresponding N-hydroxy-6-aminochrysene (N-OH-6-AC)
(Figure 1). Either N-OH-6-AC or the resultant nitrenium
Received: October 21, 2019
Published: January 6, 2020
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
© XXXX American Chemical Society
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
A

Chemical Research in Toxicology
pubs.acs.org/crt
Article
Figure 1. Metabolic activation of 6-NC and DNA adducts formed via the nitroreduction pathway.
tumorigenesis.20,21 A large fraction of mutations in most
human cancers involve mutations in the G:C base pairs of
critical genes like p53, and a relatively smaller fraction of
mutations occur at the A:T base pairs.22,23 Indeed, carcinogen-
induced mutational signatures also predominantly involve G:C
base pairs.24,25 Even so, mutations at A:T base pairs occur.26
An example of a nitroaromatic compound-derived mutation at
the A:T base pair is aristolochic acid (AA), which forms N6-dA
adducts.27 Mutational spectra of AA are dominated by A:T→
T:A transversions in the p53 gene in urothelial tumors.28,29
An in vitro repair study by El-Bayoumy and co-workers
showed that the efficiency of nucleotide excision repair (NER)
of the C8-dA adduct N-(dA-8-yl)-6-AC is ∼8 times lower than
that of the C8-dG adduct, N-(dG-8-yl)-6-AC.30 In fact, N-(dA-
8-yl)-6-AC was estimated to be more resistant to NER than all
other adducts formed by 6-NC and benzo[a]pyrene diol
epoxide.30 A DNA adduct that is repaired slowly is a significant
health concern. However, the mutagenicity of this adduct has
not yet been reported, which is a goal of our work. The prior
synthesis of this adduct was accomplished by treating an 11-
mer oligonucleotide containing a single adenine as the only
purine with N-hydroxy-6-aminochrysene (N-OH-6-AC) fol-
lowed by HPLC purification.30 In order to synthesize the
adduct in any desired DNA sequence, we decided to employ a
total synthesis approach. While many C8-dG adducts with
different polycyclic aromatic amines and NO2-PAHs have been
synthesized and incorporated in DNA by total synthesis,31−37
there are only a few reports on the synthesis of dA adducts.
Notably, syntheses of several PAH-derived DNA adducts at the
6 position of dA have been accomplished,33,38−40 but only
Meier and co-workers have incorporated C8-arylamine-
modified dA adducts in DNA by total synthesis.41,42 They
used the Pd-catalyzed Buchwald−Hartwig C−N bond-forming
strategy to incorporate phenylamine derivatives and 4-amino-
biphenyl at the C8 position of dA. To our knowledge, no other
amino-PAH has been incorporated at the C8 position of dA. In
this article, we report the synthesis of N-(dA-8-yl)-6-AC
phosphoramidite and its incorporation in DNA. We also report
its viability and mutagenicity in E. coli.
MATERIALS AND METHODS
■
All reagents were purchased from commercial chemical suppliers and
used without further purification unless otherwise noted. NMR
spectra were recorded on a Bruker Advance 400 MHz instrument. All
1H and 13C NMR data were referenced to the internal deuterated
solvent relative to TMS at 0 ppm. High-resolution mass spectroscopy
(HRMS) was performed on a QStar Elite electrospray/nanospray
quadrupole time-of-flight mass spectrometer (AB Sciex) in positive
ionization mode, and the data was analyzed using MassLynx. Flash
chromatography was performed on a Teledyne ISCO CombiFlash Rf
automated MPLC chromatography system with silica or aluminum
oxide columns. Circular dichroism (CD) spectroscopy was performed
on a Chirascan V100 spectrometer. Modified oligonucleotides were
synthesized on an ABI DNA synthesizer.
8-Bromo-2′-deoxyadenosine and 8-Bromo-3′,5′-O-bis(tert-
butyldimethylsilyl)-2′-deoxyadenosine (4). These compounds
were prepared as previously reported.42
8-Bromo-3′,5′-O-bis(tert-butyldimethylsilyl)-N6-dimethoxy-
trityl-2′-deoxyadenosine (5). 8-Bromo-3′,5′-O-bis(tert-butyldime-
thylsilyl)-2′-deoxyadenosine (4) (9.8 g, 17.442 mmol) and 4-
dimethylaminopyrine (42.2 mg, 0.3488 mmol) were dissolved in 20
mL of anhydrous pyridine. 4,4′-Dimethoxytrityl chloride (8.9 g, 26.24
mmol) was added to the solution at room temperature. The reaction
mixture was then allowed to stir at room temperature for 18 h. After
this time, thin-layer chromatography (TLC) analysis indicated
complete consumption of 4. The reaction mixture was concentrated
in vacuo, and the crude reaction mixture was purified by Al2O3 column
B
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
chromatography with a step gradient of 0−12% ethyl acetate (EtOAc)
in hexanes. The product was isolated as a foamy white solid (13.83 g,
92%).
85.60, 72.43, 70.31, 63.02, 55.16, 39.79, 25.84, 25.65, 18.24, 18.07,
−4.51, −4.72, −5.71, −5.76.
8N-(6-Aminochrysene)-3′,5′-O -bis(tert-butyldimethylsilyl)-
2′-deoxyadenosine (6a). Method 1. Fully protected coupled
substrate, 8N-(6-aminochrysene)-3′,5′-O-bis(tert-butyldimethylsilyl)-
N6-dimethoxytrityl-2′-deoxyadenosine (6b) (2.0 g, 1.95 mmol) was
dissolved in 5.0 mL of dichloromethane, and to this was added 1 M
zinc bromide solution in methanol:dichloromethane (1:1) (6 mL).
The reaction was stirred for 0.5 h and monitored for completion by
TLC (90/10 DCM/MeOH, v/v). The reaction was quenched with
aqueous sodium bicarbonate and extracted with dichloromethane.
The organic layer was combined and dried with sodium sulfate. The
solvent was evaporated under reduced pressure, and crude product
was purified by silica gel flash column chromatography with a step
gradient of 0−10% methanol in dichloromethane to afford compound
6 as a beige powder (1.05 g, 75%).
HRMS (ESI+): m/z calcd for C43H58BrN5O5Si2 [M+H]+,
860.3238; found, 860.3245.
1H NMR (400 MHz, CDCl3): δ 7.97 (s, 1H), 7.46−7.06 (m, 8H),
6.94−6.66 (m, 5H), 6.34 (t, J = 6.7 Hz, 1H), 4.98−4.76 (m, 1H),
4.05−3.84 (m, 2H), 3.80 (s, 7H), 3.77−3.52 (m, 2H), 2.22 (ddd, J =
13.0, 7.0, 4.3 Hz, 1H), 0.95 (s, 9H), 0.83 (s, 9H), 0.15 (s, 6H), −0.00
(s, 3H), −0.06 (s, 3H).
13C NMR (101 MHz, CDCl3): δ 158.53, 158.41, 158.32, 152.99,
152.90, 152.56, 151.96, 151.83, 149.83, 149.52, 145.32, 145.14,
144.90, 142.33, 138.29, 137.37, 136.37, 136.11, 130.26, 130.15,
130.12, 128.83, 128.31, 128.23, 127.88, 127.76, 127.72, 126.82,
126.79, 120.05, 113.20, 113.16, 113.05, 110.22, 108.06, 87.70, 86.49,
86.24, 85.01, 72.18, 72.12, 70.66, 62.50, 59.01, 55.21, 36.89, 25.98,
25.90, 25.78, 25.74, 18.36, 18.08, −4.63, −4.68, −5.39, −5.45.
General Procedure for Optimization of Buchwald−Hartwig
Cross-Coupling Reaction. 8-Bromo-3′,5′-O-bis(tert-butyldimethyl-
silyl)-2′-deoxyadenosine (4) (0.098 g, 0.1745 mmol) or 8-bromo-
3′,5′-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2′-deoxyade-
nosine (5) (0.150 g, 0.1745 mmol), 6-aminochrysene (0.064 mg,
0.263 mmol), Pd(0) catalyst (4 mol%), and rac-2,2′-bis-
(diphenylphosphino)-1,1′-binaphthyl (BINAP) (12.5 mg, 0.02
mmol) were suspended in 5 mL of anhydrous solvent (see Table
1). The solution was degassed by purging with argon for 1 h. The
reaction flask was then heated under argon at 85−100 °C for 1 h.
After this time sodium tert-butoxide (NaOtBu) or cesium carbonate
(Cs2CO3) (0.263 mmol) was added and the reaction stirred at 85−
100 °C for an additional 1−2 h. After this time, the reaction was
monitored via TLC for consumption of compound 4 or 5. The
reaction was then cooled, diluted with CH2Cl2, and filtered through
Celite. The filtrate was allowed to evaporate in vacuo. For substrate 4,
the crude mixture was purified on silica gel column chromatography
with a step gradient of 0−5% methanol in dichloromethane. For
substrate 5, the crude mixture was purified on Al2O3 column
chromatography with a step gradient of 0−10% EtOAc in hexanes as
the mobile phase.
Method 2. 8-Bromo-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxy-
adenosine (4) (0.098 g, 0.1745 mmol), 6-aminochrysene (0.064 mg,
0.263 mmol), tris(dibenzylideneacetone)dipalladium ((Pd2(dba)3)
(6.4 mg, 0.007 mmol), and rac-2,2′-bis(diphenylphosphino)-1,1′-
binaphthyl (BINAP) (12.5 mg, 0.02 mmol) were suspended in 8 mL
of dimethoxyethane (DME). The solution was degassed by purging
with argon for 1 h. The reaction flask was then heated under argon at
85 °C for 1 h. After this time cesium carbonate (Cs2CO3) (86 mg,
0.263 mmol) was added and the reaction stirred at 85 °C for an 1 h.
The reaction was cooled, diluted with dichloromethane, and filtered
through Celite. The filtrate was allowed to evaporate under reduced
pressure. The crude mixture was purified by silica gel column
chromatography with a step gradient of 0−5% methanol in
dichloromethane. The product 6a was isolated as a beige powder
(71 mg, 56%).
HRMS (ESI+): m/z calcd C40H52N6O3Si2 [M+H]+, 721.37; found,
721.3729.
1H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.86 (d, J = 8.4 Hz,
1H), 8.74 (dd, J = 23.0, 8.6 Hz, 3H), 8.42 (s, 1H), 8.25 (d, J = 1.9 Hz,
1H), 8.16 (d, J = 7.9 Hz, 1H), 8.00 (t, J = 9.4 Hz, 3H), 7.81−7.60 (m,
5H), 6.64 (dd, J = 8.4, 5.4 Hz, 1H), 5.30 (d, J = 15.9 Hz, 1H), 4.67
(dd, J = 5.9, 2.9 Hz, 1H), 4.22 (d, J = 2.9 Hz, 1H), 4.05 (dd, J = 11.5,
3.0 Hz, 1H), 3.88 (dd, J = 11.5, 2.8 Hz, 1H), 3.12 (dt, J = 14.0, 7.1
Hz, 1H), 2.54−2.40 (m, 1H), 0.98 (s, 9H), 0.56 (s, 9H), 0.17 (d, J =
8.7 Hz, 6H), −0.24 (s, 3H), −0.33 (s, 3H).
8N-(6-Aminochrysene)-3′,5′-O-bis(tert-butyldimethylsilyl)-
N6-dimethoxytrityl-2′-deoxyadenosine (6b). 8-Bromo-3′,5′-O-
bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2′-deoxyadenosine (5)
(0.595 g, 0.693 mmol), 6-aminochrysene (0.250 g, 1.04 mmol),
palladium(II) acetate (Pd(OAc)2) (6 mg, 0.0276 mmol), and rac-
2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (50 mg, 0.080
mmol) were suspended in 10 mL of anhydrous toluene. The solution
was degassed by purging with argon for 1 h. The reaction flask was
then heated under argon at 90 °C for 1 h. After this time cesium
carbonate (Cs2CO3) (0.366 g, 1.04 mmol) was added and the
reaction stirred at 100 °C for an additional 1 h. The reaction was
cooled, diluted with ether, and filtered through Celite. The filtrate was
allowed to evaporate under reduced pressure. The crude mixture was
purified by aluminum oxide column chromatography with a step
gradient of 0−10% EtOAc in hexanes as the mobile phase. The
product was isolated as a dark green solid (0.603 g, 85%).
HRMS (ESI+): m/z calcd C61H70N6O5Si2 [M+H]+, 1023.5025;
found, 1023.5056.
13C NMR (101 MHz, CDCl3): δ 158.53, 158.32, 152.99, 151.96,
145.32, 142.33, 137.37, 136.11, 130.15, 130.12, 128.83, 128.23,
127.88, 127.76, 126.82, 126.79, 120.05, 113.20, 113.16, 113.05,
110.22, 108.06, 87.70, 85.01, 72.12, 70.66, 62.50, 59.01, 55.21, 36.89,
25.90, 25.78, 25.74, 18.36, 18.08, −4.63, −4.68, −5.39, −5.45.
8N-(Aminochrysene)-2′-deoxyadenosine (3). 8N-(6-Amino-
chrysene)-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxyadenosine (6a)
(200 mg, 0.2772 mmol) was dissolved in 3 mL of anhydrous
tetrahydrofuran. To this was added 0.416 mL of 1 M tetrabuty-
lammonium fluoride in tetrahydrofuran, and the reaction was allowed
to stir for 24 h under N2. After this time, the solvent was evaporated
under reduced pressure, and the crude product was purified by silica
gel column chromatography with methanol (0−5%) in dichloro-
methane. The product was isolated as a light brown solid (116 mg,
85%).
MS (ESI+): m/z calcd C28H24N6O3 [M+H]+, 493.53; found,
493.534.
1H NMR (400 MHz, CDCl3): δ 9.60 (s, 1H), 8.88 (d, J = 8.2 Hz,
1H), 8.74 (d, J = 8.3 Hz, 1H), 8.70 (d, J = 9.2 Hz, 1H), 8.61 (s, 1H),
8.16 (dd, J = 8.1, 1.5 Hz, 1H), 8.03−7.92 (m, 3H), 7.81−7.54 (m,
4H), 7.42−7.35 (m, 2H), 7.33−7.29 (m, 2H), 7.28−7.17 (m, 5H),
6.85−6.70 (m, 4H), 6.65 (s, 1H), 6.53 (t, J = 7.8, 5.4 Hz, 1H), 4.23
(q, J = 3.3 Hz, 1H), 3.99 (dd, J = 11.4, 3.6 Hz, 1H), 3.91−3.80 (m,
1H), 3.77 (s, 6H), 3.21 (ddd, J = 13.4, 7.8, 5.9 Hz, 1H), 2.51 (ddd, J
= 13.0, 5.5, 2.9 Hz, 1H), 0.96 (s, 8H), 0.61 (s, 8H), 0.16 (d, J = 4.6
Hz, 6H), −0.18 (s, 3H), −0.30 (s, 3H).
1H NMR (400 MHz, DMSO-d6): δ 9.32 (s, 1H), 9.01 (d, J = 8.4
Hz, 1H), 8.89 (d, J = 7.4 Hz, 2H), 8.84 (d, J = 8.2 Hz, 3H), 8.19 (d, J
= 8.2 Hz, 1H), 8.10 (t, J = 6.8 Hz, 2H), 8.02 (s, 1H), 7.84−7.66 (m,
4H), 4.14−3.99 (m, 1H), 3.81 (d, J = 11.9 Hz, 1H), 3.10 (td, J = 15.5,
14.4, 8.3 Hz, 1H), 2.30 (dt, J = 12.9, 6.9 Hz, 1H).
13C NMR (101 MHz, DMSO-d6): δ 153.75, 150.12, 150.02,
149.66, 135.98, 132.38, 131.56, 130.00, 128.90, 128.53, 127.64,
127.43, 127.24, 127.12, 126.32, 124.26, 124.18, 124.04, 121.74,
117.26, 116.87, 88.27, 84.32, 71.90, 62.20, 58.00, 55.38, 38.79.
N6-Benzoyl-8N-(aminochrysene)-2′-deoxyadenosine (7).
8N-(6-Aminochrysene)-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxya-
denosine (6a) (445 mg, 0.617 mmol) was dissolved in anhydrous
13C NMR (101 MHz, CDCl3): δ 158.07, 151.59, 149.54, 149.20,
145.89, 138.18, 133.40, 132.49, 131.49, 130.09, 128.87, 128.83,
128.43, 127.73, 126.82, 126.70, 126.54, 126.51, 126.30, 126.11,
125.33, 123.99, 123.53, 121.38, 121.01, 118.71, 113.20, 113.04, 88.35,
C
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
a
Scheme 1. Preparation of Protected Br-dA
a
Reagents and conditions: (a) Br2, NaOAc buffer (pH 5.4), 18 h, room temperature (81%); (b) TBDMS-Cl, imidazole, pyridine, 16 h, room
temperature (95%); (c) DMT-Cl, 4-DMAP, pyridine, 18 h, room temperature (92%).
pyridine (5 mL) under an atmosphere of nitrogen, and benzoyl
chloride (0.108 mL, 0.9255) was added dropwise. The reaction
mixture was stirred for 4 h at room temperature. It was then diluted
with dichloromethane (10 mL) and washed with saturated sodium
hydrogen carbonate solution, and the aqueous layer was extracted
twice with dichloromethane. After removal of the dichloromethane
under reduced pressure, morpholine (0.134 mL, 1.5425 mmol) was
added, and the resulting mixture was stirred at room temperature for
2 h. After this time, the reaction mixture was diluted with
dichloromethane (10 mL) and then washed twice with 0.5 M sodium
dihydrogen phosphate solution. The aqueous layer was extracted
three times with dichloromethane. After complete removal of the
dichloromethane under reduced pressure, 100 mg (0.1386 mmol) of
crude product was dissolved in 3 mL of anhydrous tetrahydrofuran.
Next, 0.416 mL of 1 M tetrabutylammonium fluoride in
tetrahydrofuran was added, and the reaction was allowed to stir for
24 h under N2. After this time, the solvent was evaporated under
reduced pressure, and the crude product was purified via silica gel
column chromatography with methanol (0−10%) in dichloro-
methane. The product was isolated as a yellow solid (70 mg, 85%).
HRMS (ESI+): m/z calcd for C35H28N6O4 [M+H]+, 597.2250;
found, 597.2234 and [+Na]+.
7.33−7.27 (m, 2H), 7.27−7.20 (m, 1H), 7.13−7.07 (m, 4H), 6.87−
6.81 (m, 4H), 6.60 (ddd, J = 4.0, 2.4, 0.9 Hz, 1H), 6.30 (s, 1H),
4.23−4.18 (m, 2H), 3.82 (s, 6H), 3.68 (qd, J = 12.0, 3.1 Hz, 2H),
2.56 (dt, J = 12.5, 3.3 Hz, 1H), 2.31 (dt, J = 12.3, 5.1 Hz, 1H), 1.91 (s,
1H).
13C NMR (126 MHz, CDCl3): δ 165.57, 165.06, 158.21, 153.17,
152.09, 151.83, 149.38, 144.96, 144.53, 143.84, 136.17, 135.10,
135.01, 134.11, 132.71, 132.45, 132.10, 131.22, 130.09, 129.83,
129.77, 129.33, 128.49, 128.38, 128.24, 128.15, 127.68, 127.64,
127.01, 126.80, 126.73, 126.41, 125.91, 125.50, 124.80, 123.68,
123.51, 123.13, 122.01, 121.90, 121.10, 120.62, 115.11, 112.85,
110.01, 86.81, 86.48, 85.69, 71.79, 67.62, 63.37, 63.30, 54.93, 53.49,
53.07, 53.04, 53.02, 53.00, 52.93, 52.85, 52.83, 52.81, 52.79, 52.77,
40.96, 39.45, 0.01.
N6-Benzoyl-8N-(6-aminochrysene)-5′-O-dimethoxytrityl-2′-
deoxyadenosine-3′-O-(cyanoethyl-N,N′-diisopropylphosphor-
amidite (8). This reaction was performed in a glovebag. N6-Benzoyl-
8N-(6-aminochrysene)-5′-O-dimethoxytrityl-2′-deoxyadenosine
(0.400 g, 0.444 mmol) and N,N-diisopropylethylamine (0.155 mL,
0.888 mmol) were dissolved in anhydrous dichloromethane (6 mL)
under argon. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite
(0.105 mL, 0.444 mmol) was added and the reaction mixture stirred
for 15 min. After this time, additional N,N-diisopropylethylamine
(0.155 mL, 0.888 mmol) was added to the solution and the reaction
stirred for an additional 0.5 h. After this time, the reaction mixture
was diluted with dichloromethane (20 mL) and saturated sodium
bicarbonate (25 mL). The organic layer was retained, and the
aqueous layer was extracted twice with dichloromethane (2 × 15 mL).
The organic layers were combined, washed with brine, and dried over
sodium sulfate. The solvent was concentrated under reduced pressure
(2 mL). The solution was added dropwise to a stirring solution of 220
mL of hexanes. The resulting precipitate was collected and further
purified on basified silica gel flash chromatography with a step
gradient of 0−5% methanol in dichloromethane containing 1%
triethylamine. The product was isolated as a yellow solid (0.297 g,
61%).
1H NMR (300 MHz, DMSO-d6): δ 10.89 (s, 1H), 9.74 (s, 1H),
9.61 (s, 1H), 8.99 (d, J = 7.8 Hz, 1H), 8.83 (d, J = 8.9 Hz, 2H), 8.58
(s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 6.6 Hz, 5H), 7.79 (s,
1H), 7.59 (dt, J = 14.5, 7.4 Hz, 1H), 7.43 (dt, J = 14.4, 7.6 Hz, 3H),
6.82 (t, J = 7.1 Hz, 1H), 5.48 (d, J = 4.2 Hz, 2H), 4.57 (s, 1H), 4.12
(s, 1H), 3.91−3.59 (m, 3H), 3.27−2.97 (m, 1H), 2.42 (dd, J = 12.9,
6.0 Hz, 1H).
13C NMR (75 MHz, DMSO-d6): δ 151.51, 148.95, 145.54, 134.07,
134.00, 132.54, 132.42, 131.26, 130.18, 128.84, 128.75, 128.36,
127.60, 127.35, 127.20, 127.05, 126.96, 125.69, 124.33, 124.06,
123.33, 121.64, 115.19, 88.45, 84.85, 71.77, 62.02.
N6-Benzoyl-8N-(6-aminochrysene)-5′-O-dimethoxytrityl-2′-
deoxyadenosine. The N6-benzoylated C8-arylamine-dA adduct (7)
(0.295 g, 0.494 mmol) was dissolved in anhydrous pyridine (6 mL)
under a nitrogen atmosphere, and 4,4′-dimethoxytrityl chloride
(0.177 g, 0.522 mmol) and silver nitrate (0.089 g, 0.522 mmol)
were added. The mixture was stirred at room temperature until the
reaction was complete (18 h). The reaction mixture was then diluted
with dichloromethane (10 mL) and washed with saturated sodium
hydrogen carbonate solution and brine. The aqueous layer was
extracted twice with dichloromethane. The organic layers were
combined, dried over sodium sulfate, and filtered, and the solvent was
removed under reduced pressure. The residue was purified by flash
chromatography on silica gel, eluting with 0−3% methanol in
dichloromethane to afford the desired DMT-protected 7 as a yellow
foam (328 mg, 74%).
HRMS (ESI+): m/z calcd C65H63N8O7P [M+H]+, 1099.4636;
found, 1099.4607.
31P NMR (162 MHz, CD2Cl2): δ 149.06, 149.01, 148.57, 148.51.
Site-Specific Synthesis of Oligodeoxynucleotides Contain-
ing N-(dA-8-yl)-6-AC Adduct. Phosphoramidite 8 was used to
synthesize modified oligodeoxynucleotides as per the manufacturer’s
instruction. However, the time of the coupling step was extended to
15 min for the incorporation of modified phosphoramidite with total
coupling efficiencies of >95%. The unmodified and complementary
oligodeoxynucleotide sequences were purchased from Integrated
DNA Technologies, Inc. (Coralville, Iowa, USA). The modified
oligonucleotides were purified by reverse-phase HPLC followed by
denaturing PAGE. The homogeneity of the purified modified and
unmodified (control) oligodeoxynucleotides was confirmed via
phosphorylation with T4 polynucleotide kinase in the presence of
[γ32P]adenosine triphosphate and subsequent PAGE analysis (see SI,
Figure S27). The modified oligodeoxynucleotides were further
characterized by ES mass spectrometry. HRMS data are given in
Table 2.
HRMS (ESI+): m/z calcd C56H46N6O6 [M+H]+, 899.3552; found,
899.3532.
1H NMR (500 MHz, chloroform-d): δ 9.31 (s, 1H), 8.76 (dd, J =
7.5, 1.3 Hz, 1H), 8.72−8.73 (m, 1H), 8.44 (s, 1H), 8.23 (s, 1H), 8.16
(dd, J = 7.8, 1.3 Hz, 1H), 8.05−8.03 (m, 2H), 8.00−7.94 (m, 1H),
7.89 (d, J = 9.2 Hz, 1H), 7.72−7.66 (m, 2H), 7.66−7.62 (m, 2H),
7.54−7.42 (m, 3H), 7.42−7.38 (m, 2H), 7.37 (d, J = 1.4 Hz, 1H),
D
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
a
Table 1. Optimization of Buchwald−Hartwig Cross-Coupling Reaction
entry
X
catalyst (4 mol%)
base (1.5 equiv)
ligand (12 mol%)
solvent
temp (°C)
yield (%)
1
2
3
4
5
6
7
8
H
H
H
H
DMT
DMT
DMT
DMT
Pd2(dba)3
Pd(OAc)2
Pd2(dba)3
Pd(OAc)2
Pd2(dba)3
Pd(OAc)2
Pd2(dba)3
Pd(OAc)2
NaOtBu
Cs2CO3
NaOtBu
Cs2CO3
NaOtBu
Cs2CO3
NaOtBu
Cs2CO3
BINAP
BINAP
BINAP
BINAP
BINAP
BINAP
BINAP
BINAP
toluene
toluene
DME
100
100
85
45
56
52
42
75
85
68
61
DME
85
toluene
toluene
DME
100
100
85
DME
85
a
All reactions were carried out for 3 h.
a
Scheme 2. Synthesis of 5′-DMT-Protected 3′-Phosphoramidite 8
a
Reagents and conditions: (a) 1 M ZnBr2 in 1:1 MeOH:CH2Cl2 solution, 0.5 h, 75%; (b) 1 M TBAF in THF, RT, 24 h, 85%; (c) benzoyl chloride
(BzCl), pyridine, RT followed by treatment with morpholine, 2 h; (d) 1 M TBAF in THF, RT, 24 h, 85%; (e) 4,4′-dimethoxytrityl chloride (DMT-
Cl), pyridine, AgNO3, RT, 18 h, 74%; (f) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIEA, CH2Cl2, RT, 1 h, 61%.
Circular Dichroism Studies. The concentration of oligonucleo-
tides was determined using a NanoDrop 2000 spectrophotometer.
Equal amounts of two complementary strands (5 nmol) were
dissolved in buffer [1 mL, NaH2PO4 buffer (10 mM), NaCl (140
mM), EDTA (1 mM), pH 6.6], heated to 70 °C, and then slowly
allowed to cool to room temperature to enable annealing of the two
strands. CD measurements were carried out at 25 °C, and samples
were scanned from 400 to 220 nm at 0.5 nm intervals averaged over 5
s.
containing plasmid and an unmodified control pMS2 plasmid and
their replication in E. coli AB 1157 essentially followed the protocol
described in detail in refs 43 and 44. SOS induction was carried out
with 50 J/m2 of UV light (254 nm). Oligonucleotide hybridization
also was performed as reported.45,46 Lesion bypass efficiency was
calculated by comparing the transformation efficiency of the N-(dA-8-
yl)-6-AC construct with that of the control, whereas mutation
frequency (MF) was calculated based on hybridization data and
sequence analysis.
Construction of Adduct-Containing Plasmid, SOS Induc-
tion, and Transformation in E. coli. Construction of the adduct-
E
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
Figure 2. ROESY-NMR spectra of N-(dA-8-yl)-6-AC adduct 3 in DMSO-d6 at 300 K.
protected substrate in the Buchwald−Hartwig cross-coupling
reactions can be attributed to the increased solubility of the
substrate in toluene (and DME), facilitated by the nonpolar
DMT group.
RESULTS AND DISCUSSION
■
Protected Br-dA (4) was prepared by a standard method of
selective bromination at the C8 position of dA using Br2 in
acetate buffer (pH 5.4) followed by TBDMS protection of the
3′ and 5′ hydroxyl groups (Scheme 1). Following Meier’s
strategy for coupling arylamines with dA,41,42 we first carried
out Buchwald−Hartwig cross-coupling with compound 4 and
6-aminochrysene (6-AC) using Pd2(dba)3 or Pd(OAc)2
catalyst with BINAP and either NaOtBu or Cs2CO3 as the
base in toluene or DME. The C−N coupling worked in each
case, but the yield remained between 42 and 56% (Table 1).
We found that 12 mol% of BINAP was optimal for these
reactions, and higher mol% caused partial degradation of the
product. Each reaction shown here was carried out for 3 h.
Longer reaction times (up to 24 h) led to significant tailing on
TLC for reactions carried out at 100 °C in toluene, which is
usually indicative of degradation of the product. In 1,2-DME
(performed at 85 °C) this tailing was not observed, and the
reactions were allowed to run overnight. However, longer
reactions times did not result in improvement in reaction
yields.
With the optimized conditions to access coupled product
6b, the DMT group on N6-excocyclic amine was deprotected
using a 1 M solution of ZnBr2 to furnish 6a (Scheme 2). The
N6-amine functionality was then reprotected with a base-labile
benzoyl group using benzoyl chloride in pyridine followed by
treatment with morpholine to afford N6-benzoylated product,
and desilylation of the 5′- and 3′-hydroxyls was achieved using
tetrabutylammonium fluoride (TBAF) in THF to furnish 7.
The 5′-hydroxyl was protected with the acid-labile DMT group
and used to prepare the 3′-phosphoramidite using 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite to give the
phosphoramidite monomer 8 (Scheme 2).
Syn/Anti Conformational Preference of N-(dA-8-yl)-6-
AC Adduct 3. NMR data on C8-arylamine-dA adducts
showed that, with a single phenyl ring, the preferred
conformation of the N-glyosidic bond is anti, but addition of
an N-acetyl group replacing the amino hydrogen rotates it to
syn conformation.42 To determine syn or anti preference of N-
(dA-8-yl)-6-AC, its conformation was investigated using
ROESY-NMR spectroscopy. Figure 2 shows a cross-peak
between the H1′ proton of the sugar and 6-AC amine tethered
to the C8 site of dA, indicating a preference for syn
conformation. Furthermore, no cross-peaks were observed
between the H2 proton on the nucleobase and the H1′ proton
on the sugar, typically observed for the anti conformation.
These results indicate a preference for N-(dA-8-yl)-6-AC to
exist in the syn conformation of the N-glyosidic bond, which
was also reported for other bulky C8-arylamine-modified
nucleosides.48,49
For the Pd-catalyzed coupling of protected dG with
polyaromatic amines, Gillet and Scharer showed that N -
2
̈
DMT-protected dG provides an excellent yield of the adduct,34
which we have used successfully for the synthesis of dG-1-
aminopyrene adduct.47 So, to improve the coupling efficiency,
the N6 exocyclic amine was protected using 4,4′-dimethoxy-
trityl chloride (DMT-Cl) in pyridine with catalytic amounts of
4-dimethylaminopyridine (DMAP), and then the N6DMT-
protected Br-dA nucleoside 5 was subjected to the Pd-
catalyzed coupling. We are pleased to report better coupling
efficiency for each reaction, and the best coupling yield of 85%
was achieved with Pd(OAc)2/Cs2CO3/BINAP combination in
toluene at 100 °C for 3 h (Table 1). For the DMT-protected
Br-dA nucleoside 5 also longer reaction time did not improve
the yield. We believe that the improved yields with N6-DMT-
Synthesis of Oligodeoxynucleotides of N-(dA-8-yl)-6-
AC Adduct. The protected N-(dA-8-yl)-6-AC phosphorami-
dite 8 was used to synthesize 12- and 15-mer oligonucleotides,
F
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
and the coupling efficiency was more than 95% for the
modified dA in each case. The oligonucleotides were
deprotected at 55 °C with ammonium hydroxide for 24 h.
During deprotection, 0.25 M 2-mercaptoethanol was added to
avoid oxidative degradation. The modified oligodeoxynucleo-
tides were purified by reverse-phase HPLC followed by
polyacrylamide gel electrophoresis (PAGE) and characterized
by high-resolution electrospray ionization mass spectrometry
in negative mode (Table 2).
Mutational Analyses of N-(dA-8-yl)-6-AC in E. coli.
The DNA sequence of the 15-mer was chosen from TP53 gene
codon 129−133, because crops contaminated with another
nitroaromatic carcinogen aristolochic acid (AA) caused A→T
mutation in codon 131 in patients with urothelial tumors.23,50
While many polyaromatic amine and nitroaromatic com-
pounds form adducts at the C8 position of dG, which induce
frameshifts and G→T transversions as the dominant
mutations,43,51,52 the major AA adducts are formed at the N6
position of dA.27 The 15-mer oligonucleotide (5′-
GCCCTCAA*CAAGATG-3′) was incorporated into a
scaffolded single-stranded pMS2 plasmid as reported for the
construction of other DNA lesions, after which the scaffold was
enzymatically removed.53,54 An unmodified control plasmid
was also prepared using the same approach. The N-(dA-8-yl)-
6-AC-containing and control plasmids were then replicated in
uninduced and SOS-induced E. coli AB1157 cells. In
uninduced cells, the number of progeny colonies from the
adduct-containing construct was reduced to ∼60% to that of
the control (Figure 4A and SI, Table S1). This suggests that
though replication was inhibited, N-(dA-8-yl)-6-AC is less
toxic than the C8 dG adduct of 1-nitropyrene, which gives less
than 30% progeny.52,55 Upon induction of SOS, viability of the
adducted genome increased to nearly 90%, indicating
significantly more facile translesion synthesis (TLS) of N-
(dA-8-yl)-6-AC by the SOS proteins (Figure 4A and SI, Table
S1). It would be of interest to determine in the future which
SOS proteins participate in the increased TLS of this adduct by
using E. coli strains with appropriately knocked out TLS
polymerase(s).56
Table 2. HPLC-ESI HRMS (Negative Mode) Data for
Modified and Unmodified (Control) Oligodeoxynucleotides
m/z (Da) (charge)
a
oligodeoxynucleotides
calculated
observed
5′-GTGCAT GTT TGT-3′
5′-GTGCA*T GTT TGT-3′
5′-GCCCTCAACAAGATG-3′
5′-GCCCTCAA*CAAGATG-3′
1225.8680 (−3)
1306.3212 (−3)
1134.8941 (−4)
1195.2252 (−4)
1225.9200 (−3)
1306.2352 (−3)
1134.9442 (−4)
1195.2252 (−4)
a
A* represents N-(dA-8-yl)-6-AC.
Circular Dichroism (CD) Studies. The 15-mer and 12-
mer unmodified and N-(dA-8-yl)-6-AC-modified oligodeox-
ynucleotides were allowed to anneal to their complementary
strand, and each of the resultant duplexes was evaluated on a
CD spectrometer (Chirascan V100). As shown in Figure 3, the
CD spectra confirm overall B-type DNA conformation of the
unmodified control duplex and the N-(dA-8-yl)-6-AC-contain-
ing duplex. For the control duplex a positive Cotton effect was
observed at 280 nm along with a negative one at 245 nm
typically found in B-type DNA. However, the CD curve of the
N-(dA-8-yl)-6-AC-containing duplex shows a slight deviation
in the B-type DNA helical structure of the unmodified duplex.
Nevertheless, the positive and negative Cotton effects observed
were consistent with canonical B-type DNA structures.
Without SOS, 5.2% of the replicates were mutants, which
included 2.9% targeted (i.e., mutations at the modified A) and
2.3% semi-targeted mutations (Figure 4B), the latter being
defined as mutations near the lesion site. Of the targeted
mutations, 80% were A*→G transitions. With SOS, MF
increased to 12.2%, showing 3-fold increase of the targeted
Figure 3. CD spectra of unmodified (red solid line) and modified (black solid line) 15-mer (A) and 12-mer (B) oligonucleotides (sequence shown
in Table 2) after annealing with their complementary strand. The CD spectra of the single-stranded oligonucleotides are shown in black dotted
lines.
G
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
Figure 4. (A) Bypass efficiencies in N-(dA-8-yl)-6-AC construct in E. coli (−) SOS (WT) and (+) SOS cells. The data represent the means and
standard errors of results from three independent experiments. (B) Frequencies of total targeted and semi-targeted mutations and (C) frequencies
of specific types of targeted mutations induced in progeny from N-(dA-8-yl)-6-AC construct in (−) SOS (WT) and (+) SOS E. coli cells. The data
represent the means and standard errors of results from three independent experiments. *P < 0.05; ***P < 0.001. The P-values were calculated by
using unpaired two-tailed Student’s t-test.
mutations to 9% frequency, whereas semi-targeted mutation
increased only marginally to 3.2% (Figure 4C and SI, Table
S1). Although A*→G was also the prevalent (55%) targeted
mutation with SOS, nearly half as many A*→C was detected.
Although the predominant conformation of N-(dA-8-yl)-6-
AC is syn, it is not known if the same conformation is
maintained in DNA. But many bulky polyaromatic moieties at
the C8 position of dG in DNA predominantly rotate the base
to syn conformation.49 It was speculated that syn conformation
of an adduct may be more repair-prone than others, but
mutagenic relevance to these conformational preferences is
significantly more complex.49 Additional structural studies will
be necessary to determine if the conformation of N-(dA-8-yl)-
6-AC played a role in the observed mutagenesis. Even so, it is
likely that the TLS polymerases are involved in the error-prone
bypass of N-(dA-8-yl)-6-AC, as reflected in an increase in the
targeted MF with SOS.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.chemrestox.9b00429.
NMR and MS of the synthesized compounds, an
autoradiogram of denaturing polyacrylamide gel electro-
phoresis of the modified oligonucleotide in comparison
to their unmodified counterparts, and a table of TLS
efficiency and MF (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Ashis K. Basu − University of Connecticut, Storrs,
Connecticut; orcid.org/0000-0001-7951-4984;
Phone: (860) 486-3965; Email: ashis.basu@uconn.edu
Other Author
In conclusion, an efficient strategy for the synthesis of N-
(dA-8-yl)-6-AC has been developed. The 6-aminochrysene
moiety was introduced at the C8 position of dA using
Buchwald−Hartwig palladium-catalyzed cross-coupling chem-
istry. This optimized strategy provided efficient and convenient
access to this adduct. Using ROESY-NMR spectroscopy, we
determined that the adducted purine prefers syn conformation
of its glyosidic bond of N-(dA-8-yl)-6-AC in solution. The 3′-
phosphoramidite of N-(dA-8-yl)-6-AC was used to site-
specifically incorporate the dA-adduct into oligonucleotides
by solid-phase DNA synthesis. The CD spectra of the 15-mer
duplex were consistent with B-DNA, although it showed small
deviations from the control duplex, suggesting a less structured
helix. In terms of the biological effects of N-(dA-8-yl)-6-AC, it
stalls DNA synthesis in E. coli, but TLS increased to ∼90%
with SOS. The predominant mutation induced by N-(dA-8-
yl)-6-AC in E. coli was A→G transitions, which increased
significantly with SOS.
Brent V. Powell − University of Connecticut, Storrs,
Connecticut
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.9b00429
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
NO2-PAH, nitropolycyclic aromatic hydrocarbons; 6-NC, 6-
nitrochrysene; N-(dG-8-yl)-6-AC, N-(deoxyguanosin-8-yl)-6-
aminochrysene; 5-(dG-N2-yl)-6-AC, 5-(deoxyguanosin-N2-yl)-
6-aminochrysenene; N-(dA-8-yl)-6-AC, N-(deoxyadenosin-8-
yl)-6-aminochrysene; AA, aristolochic acid; NER, nucleotide
excision repair; PAH, polynuclear aromatic hydrocarbon;
BINAP, rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl;
PAGE, polyacrylamide gel electrophoresis; CD, circular
dichroism; MF, mutation frequency; TLS, translesion synthesis
H
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
(17) Sun, Y. W., Guttenplan, J. B., Khmelnitsky, M., Krzeminski, J.,
Boyiri, T., Amin, S., and El-Bayoumy, K. (2009) Stereoselective
metabolism of the environmental mammary carcinogen 6-nitro-
chrysene to trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene by ar-
oclor 1254-treated rat liver microsomes and their comparative
mutation profiles in a laci mammary epithelial cell line. Chem. Res.
Toxicol. 22, 1992−1997.
(18) Imaida, K., Uneyama, C., Ogasawara, H., Hayashi, S., Fukuhara,
K., Miyata, N., and Takahashi, M. (1992) Induction of colon
adenocarcinomas in CD rats and lung adenomas in ICR mice by 6-
nitrochrysene: comparison of carcinogenicity and aryl hydrocarbon
hydroxylase induction in the target organs of each species. Cancer Res.
52, 1542−1545.
(19) Boyiri, T., Guttenplan, J., Khmelnitsky, M., Kosinska, W., Lin, J.
M., Desai, D., Amin, S., Pittman, B., and El-Bayoumy, K. (2003)
Mammary carcinogenesis and molecular analysis of in vivo cII gene
mutations in the mammary tissue of female transgenic rats treated
with the environmental pollutant 6-nitrochrysene. Carcinogenesis 25,
637−643.
(20) Wogan, G. N., Hecht, S. S., Felton, J. S., Conney, A. H., and
Loeb, L. A. (2004) Environmental and chemical carcinogenesis.
Semin. Cancer Biol. 14, 473−486.
(21) Harris, C. C. (1992) Tumour suppressor genes, multistage
carcinogenesis and molecular epidemiology. IARC Sci. Publ. 116, 67−
85.
(22) Hainaut, P., Soussi, T., Shomer, B., Hollstein, M., Greenblatt,
M., Hovig, E., Harris, C. C., and Montesano, R. (1997) Database of
p53 gene somatic mutations in human tumors and cell lines: updated
compilation and future prospects. Nucleic Acids Res. 25, 151−157.
(23) Olivier, M., Hollstein, M., and Hainaut, P. (2010) TP53
mutations in human cancers: origins, consequences, and clinical use.
Cold Spring Harbor Perspect. Biol. 2, No. a001008.
(24) Reinbold, M., Luo, J. L., Nedelko, T., Jerchow, B., Murphy, M.
E., Whibley, C., Wei, Q., and Hollstein, M. (2008) Common tumour
p53 mutations in immortalized cells from Hupki mice heterozygous at
codon 72. Oncogene 27, 2788−2794.
(25) vom Brocke, J., Krais, A., Whibley, C., Hollstein, M. C., and
Schmeiser, H. H. (2008) The carcinogenic air pollutant 3-nitro-
benzanthrone induces GC to TA transversion mutations in human
p53 sequences. Mutagenesis 24, 17−23.
(26) Woodrick, J., Gupta, S., Khatkar, P., Sarangi, S., Narasimhan,
G., Trehan, A., Adhikari, S., and Roy, R. (2014) Slow repair of lipid
peroxidation-induced DNA damage at p53 mutation hotspots in
human cells caused by low turnover of a DNA glycosylase. Nucleic
Acids Res. 42, 9033−9046.
(27) Grollman, A. P., Shibutani, S., Moriya, M., Miller, F., Wu, L.,
Moll, U., Suzuki, N., Fernandes, A., Rosenquist, T., Medverec, Z.,
Jakovina, K., Brdar, B., Slade, N., Turesky, R. J., Goodenough, A. K.,
Rieger, R., Vukelic, M., and Jelakovic, B. (2007) Aristolochic acid and
the etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci.
U. S. A. 104, 12129−12134.
(28) Moriya, M., Slade, N., Brdar, B., Medverec, Z., Tomic, K.,
Jelakovic, B., Wu, L., Truong, S., Fernandes, A., and Grollman, A. P.
(2011) TP53 Mutational signature for aristolochic acid: an environ-
mental carcinogen. Int. J. Cancer 129, 1532−1536.
(29) Hashimoto, K., Bonala, R., Johnson, F., Grollman, A. P., and
Moriya, M. (2016) Y-family DNA polymerase-independent gap-filling
translesion synthesis across aristolochic acid-derived adenine adducts
in mouse cells. DNA Repair 46, 55−60.
(30) Krzeminski, J., Kropachev, K., Reeves, D., Kolbanovskiy, A.,
Kolbanovskiy, M., Chen, K. M., Sharma, A. K., Geacintov, N., Amin,
S., and El-Bayoumy, K. (2013) Adenine-DNA adduct derived from
the nitroreduction of 6-nitrochrysene is more resistant to nucleotide
excision repair than guanine-DNA adducts. Chem. Res. Toxicol. 26,
1746−1754.
(31) Santosh Laxmi, Y. R., Suzuki, N., Dasaradhi, L., Johnson, F.,
and Shibutani, S. (2002) Preparation of oligodeoxynucleotides
containing a diastereoisomer of alpha-(N(2)-2′-deoxyguanosinyl)-
REFERENCES
■
(1) Pitts, J. N., Jr., Van Cauwenberghe, K. A., Grosjean, D., Schmid,
J. P., Fitz, D. R., Belser, W. L., Knudson, G. P., and Hynds, P. M.
(1978) Atmospheric reactions of polycyclic aromatic hydrocarbons:
facile formation of mutagenic nitro derivatives. Science 202, 515−519.
(2) Rosenkranz, H. S., McCoy, E. C., Sanders, D. R., Butler, M.,
Kiriazides, D. K., and Mermelstein, R. (1980) Nitropyrenes: isolation,
identificaton, and reduction of mutagenic impurities in carbon black
and toners. Science 209, 1039−1043.
(3) Gentner, D. R., Isaacman, G., Worton, D. R., Chan, A. W.,
Dallmann, T. R., Davis, L., Liu, S., Day, D. A., Russell, L. M., Wilson,
K. R., Weber, R., Guha, A., Harley, R. A., and Goldstein, A. H. (2012)
Elucidating secondary organic aerosol from diesel and gasoline
vehicles through detailed characterization of organic carbon
emissions. Proc. Natl. Acad. Sci. U. S. A. 109, 18318−18323.
(4) Beland, F. A., and Marques, M. M. (1994) DNA adducts of
nitropolycyclic aromatic hydrocarbons. IARC Sci. Publ. 125, 229−244.
(5) Purohit, V., and Basu, A. K. (2000) Mutagenicity of
nitroaromatic compounds. Chem. Res. Toxicol. 13, 673−692.
(6) Benbrahim-Tallaa, L., Baan, R. A., Grosse, Y., Lauby-Secretan, B.,
El Ghissassi, F., Bouvard, V., Guha, N., Loomis, D., and Straif, K.
(2012) Carcinogenicity of diesel-engine and gasoline-engine exhausts
and some nitroarenes. Lancet Oncol. 13, 663−664.
(7) el-Bayoumy, K., Desai, D., Upadhyaya, P., Amin, S., and Hecht,
S. S. (1992) Comparative tumorigenicity of nitrochrysene isomers in
newborn mice. Carcinogenesis 13, 2271−2275.
(8) El-Bayoumy, K., Delcios, K. B., Heflich, R. H., Walker, R., Shiue,
G.-H., and Hecht, S. S. (1989) Mutagenicity, metabolism and DNA
adduct formation of 6-nitrochrysene in Salmonella typhimurium.
Mutagenesis 4, 235−240.
(9) el-Bayoumy, K. (1992) Environmental carcinogens that may be
involved in human breast cancer etiology. Chem. Res. Toxicol. 5, 585−
590.
(10) El-Bayoumy, K., Desai, D., Boyiri, T., Rosa, J., Krzeminski, J.,
Sharma, A. K., Pittman, B., and Amin, S. (2002) Comparative
tumorigenicity of the environmental pollutant 6-nitrochrysene and its
metabolites in the rat mammary gland. Chem. Res. Toxicol. 15, 972−
978.
(11) Boyiri, T., Leszczynska, J., Desai, D., Amin, S., Nixon, D. W.,
and El-Bayoumy, K. (2002) Metabolism and DNA binding of the
environmental pollutant 6-nitrochrysene in primary culture of human
breast cells and in cultured MCF-10A, MCF-7 and MDA-MB-435s
cell lines. Int. J. Cancer 100, 395−400.
(12) Delclos, K. B., Walker, R. P., Dooley, K. L., Fu, P. P., and
Kadlubar, F. F. (1987) Carcinogen-DNA adduct formation in the
lungs and livers of preweanling CD-1 male mice following
administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and
[3H]-1,6-dinitropyrene. Cancer Res. 47, 6272−6277.
(13) Delclos, K. B., Miller, D. W., Lay, J. O., Jr., Casciano, D. A.,
Walker, R. P., Fu, P. P., and Kadlubar, F. F. (1987) Identification of
C8-modified deoxyinosine and N2- and C8-modified deoxyguanosine
as major products of the in vitro reaction of N-hydroxy-6-
aminochrysene with DNA and the formation of these adducts in
isolated rat hepatocytes treated with 6-nitrochrysene and 6-amino-
chrysene. Carcinogenesis 8, 1703−1709.
(14) Delclos, K. B., El-Bayoumy, K., Casciano, D. A., Walker, R. P.,
Kadlubar, F. F., Hecht, S. S., Shivapurkar, N., Mandal, S., and Stoner,
G. D. (1989) Metabolic activation of 6-nitrochrysene in explants of
human bronchus and in isolated rat hepatocytes. Cancer Res. 49,
2909−2913.
(15) Chae, Y. H., Delclos, K. B., Blaydes, B., and El-Bayoumy, K.
(1996) Metabolism and DNA binding of the environmental colon
carcinogen 6-nitrochrysene in rats. Cancer Res. 56, 2052−2058.
(16) El-Bayoumy, K., Sharma, A. K., Lin, J. M., Krzeminski, J., Boyiri,
T., King, L. C., Lambert, G., Padgett, W., Nesnow, S., and Amin, S.
(2004) Identification of 5-(deoxyguanosin-N2-yl)- 1,2-dihydroxy-1,2-
dihydro-6-aminochrysene as the major DNA lesion in the mammary
gland of rats treated with the environmental pollutant 6-nitro-
chrysene. Chem. Res. Toxicol. 17, 1591−1599.
I
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
tamoxifen by phosphoramidite chemical synthesis. Chem. Res. Toxicol.
15, 218−225.
(32) Bonala, R. R., Torres, M. C., Attaluri, S., Iden, C. R., and
Johnson, F. (2005) Incorporation of N2-deoxyguanosine metabolic
adducts of 2-aminonaphthalene and 2-aminofluorene into oligomeric
DNA. Chem. Res. Toxicol. 18, 457−465.
(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dC
in a DNA duplex. Biochemistry 35, 12659−12670.
(49) Patel, D. J., Mao, B., Gu, Z., Hingerty, B. E., Gorin, A., Basu, A.
K., and Broyde, S. (1998) Nuclear magnetic resonance solution
structures of covalent aromatic amine-DNA adducts and their
mutagenic relevance. Chem. Res. Toxicol. 11, 391−407.
(50) Hranjec, T., Kovac, A., Kos, J., Mao, W., Chen, J. J., Grollman,
A. P., and Jelakovic, B. (2005) Endemic nephropathy: the case for
chronic poisoning by aristolochia. Croat. Med. J. 46, 116−125.
(51) Wood, M. L., Esteve, A., Morningstar, M. L., Kuziemko, G. M.,
and Essigmann, J. M. (1992) Genetic effects of oxidative DNA
damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and
7,8-dihydro-8-oxoadenine in Escherichia coli. Nucleic Acids Res. 20,
6023−6032.
(52) Bacolod, M. D., Krishnasamy, R., and Basu, A. K. (2000)
Mutagenicity of the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-
yl)-1-aminopyrene in Escherichia coli located in a nonrepetitive CGC
sequence. Chem. Res. Toxicol. 13, 523−528.
(53) Pande, P., Malik, C. K., Bose, A., Jasti, V. P., and Basu, A. K.
(2014) Mutational analysis of the C8-guanine adduct of the
environmental carcinogen 3-nitrobenzanthrone in human cells:
critical roles of DNA polymerases eta and kappa and Rev1 in error-
prone translesion synthesis. Biochemistry 53, 5323−5331.
(54) Bose, A., Pande, P., Jasti, V. P., Millsap, A. D., Hawkins, E. K.,
Rizzo, C. J., and Basu, A. K. (2015) DNA polymerases kappa and zeta
cooperatively perform mutagenic translesion synthesis of the C8−2′-
deoxyguanosine adduct of the dietary mutagen IQ in human cells.
Nucleic Acids Res. 43, 8340−8351.
(33) De Riccardis, F., Bonala, R. R., and Johnson, F. (1999) A
general method for the synthesis of the N2- and N6- carcinogenic
amine adducts of 2′-deoxyguanosine and 2′-deoxyadenosine. J. Am.
Chem. Soc. 121, 10453−10460.
̈
(34) Gillet, L. C., and Scharer, O. D. (2002) Preparation of C8-
amine and acetylamine adducts of 2′-deoxyguanosine suitably
protected for DNA synthesis. Org. Lett. 4, 4205−4208.
(35) Gillet, L. C., Alzeer, J., and Scharer, O. D. (2005) Site-specific
incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-
AAF) into oligonucleotides using modified ‘ultra-mild’ DNA syn-
thesis. Nucleic Acids Res. 33, 1961−1969.
(36) Chakraborti, D., Colis, L., Schneider, R., and Basu, A. K. (2003)
Synthesis of N2 2′-deoxyguanosine adducts formed by 1-nitropyrene.
Org. Lett. 5, 2861−2864.
(37) Wang, Z., and Rizzo, C. J. (2001) Synthesis of the C8-
deoxyguanosine adduct of the food mutagen IQ. Org. Lett. 3, 565−
568.
(38) Yagi, H., Frank, H., Seidel, A., and Jerina, D. M. (2008) Revised
assignment of absolute configuration of the cis- and trans-N6-
deoxyadenosine adducts at C14 of ( )-11beta,12alpha-dihydroxy-
13alpha,14alpha-epoxy-11,12,13,14-tetrahydrodibenz o[a, l]pyrene by
stereoselective synthesis. Chem. Res. Toxicol. 21, 2379−2392.
(39) Attaluri, S., Bonala, R. R., Yang, I. Y., Lukin, M. A., Wen, Y.,
Grollman, A. P., Moriya, M., Iden, C. R., and Johnson, F. (2010)
DNA adducts of aristolochic acid II: total synthesis and site-specific
mutagenesis studies in mammalian cells. Nucleic Acids Res. 38, 339−
352.
(40) Lakshman, M. K., Keeler, J. C., Hilmer, J. H., and Martin, J. Q.
(1999) Palladium-catalyzed C-N bond formation: Facile and general
synthesis of N6-aryl 2′-deoxyadenosine analogues. J. Am. Chem. Soc.
121, 6090−6091.
(55) Malia, S. A., Vyas, R. R., and Basu, A. K. (1996) Site-specific
frame-shift mutagenesis by the 1-nitropyrene-DNA adduct N-
(deoxyguanosin-8-y1)-1-aminopyrene located in the (CG)3 se-
quence: effects of SOS, proofreading, and mismatch repair.
Biochemistry 35, 4568−4577.
(56) Maslowska, K. H., Makiela-Dzbenska, K., and Fijalkowska, I. J.
(2019) The SOS system: A complex and tightly regulated response to
DNA damage. Environ. Mol. Mutagen. 60, 368−384.
(41) Jacobsen, M. I., and Meier, C. (2008) Oligonucleotides
damaged with carcinogenic aromatic amines at the C8- and N6-
position of 2′-deoxyadenosine. Nucleic Acids Symp. Ser. (Oxf.) 52,
439−440.
(42) Szombati, Z., Baerns, S., Marx, A., and Meier, C. (2012)
Synthesis of C8-arylamine-modified 2′-deoxyadenosine phosphor-
amidites and their site-specific incorporation into oligonucleotides.
ChemBioChem 13, 700−712.
(43) Hilario, P., Yan, S., Hingerty, B. E., Broyde, S., and Basu, A. K.
(2002) Comparative mutagenesis of the C8-guanine adducts of 1-
nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence.
A slipped frameshift intermediate model for dinucleotide deletion. J.
Biol. Chem. 277, 45068−45074.
(44) Weerasooriya, S., Jasti, V. P., and Basu, A. K. (2014) Replicative
bypass of abasic site in Escherichia coli and human cells: similarities
and differences. PLoS One 9, No. e107915.
(45) Watt, D. L., Utzat, C. D., Hilario, P., and Basu, A. K. (2007)
Mutagenicity of the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-
yl)-1-aminopyrene in mammalian cells. Chem. Res. Toxicol. 20, 1658−
1664.
(46) Kalam, M. A., Haraguchi, K., Chandani, S., Loechler, E. L.,
Moriya, M., Greenberg, M. M., and Basu, A. K. (2006) Genetic effects
of oxidative DNA damages: comparative mutagenesis of the imidazole
ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines
in simian kidney cells. Nucleic Acids Res. 34, 2305−2315.
(47) Colis, L. C., Chakraborti, D., Hilario, P., McCarty, C., and Basu,
A. K. (2009) Synthesis of oligonucleotides containing 2′-deoxy-
guanosine adducts of nitropyrenes. Nucleosides, Nucleotides Nucleic
Acids 28, 67−77.
(48) Mao, B., Vyas, R. R., Hingerty, B. E., Broyde, S., Basu, A. K.,
and Patel, D. J. (1996) Solution conformation of the N-
J
https://dx.doi.org/10.1021/acs.chemrestox.9b00429
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX