Analysis and Identification of 2′-Deoxyadenosine-Derived Adducts in Lung and Liver DNA of F-344 Rats Treated with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

Article abstract of DOI:10.1021/acs.chemrestox.8b00056

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are carcinogenic in animal models and are believed to play an important role in human lung carcinogenesis for cigarette smokers. Cytochrome P450-mediated metabolism of these tobacco-specific nitrosamines produces reactive species that alkylate DNA in the form of pyridyloxobutyl (POB)- or pyridylhydroxybutyl (PHB)-DNA adducts. Understanding the formation mechanism and overall levels of these adducts can potentially enhance cancer prevention methods through the identification of particularly susceptible smokers. Previous studies have identified and measured a panel of POB- and PHB-DNA base adducts of dGuo, dCyd, and Thd; however, dAdo adducts have yet to be determined. In this study, we complete this DNA adduct panel by identifying and quantifying levels of NNK- and NNAL-derived dAdo adducts in vitro and in vivo. To accomplish this, we synthesized standards for expected dAdo-derived DNA adducts and used isotope-dilution LC-ESI⁺-MS/MS to identify POB adducts formed in vitro from the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) with calf thymus DNA. Adduct levels were then quantified in lung and liver DNA of rats chronically treated with NNK or NNAL for 50 weeks using similar LC-MS detection methods. The in vitro studies identified N⁶-POB-dAdo and N¹-POB-dIno as products of the reaction of NNKOAc with DNA, which supports our proposed mechanism of formation. Though both N⁶-dAdo and N¹-dIno adducts were found in vitro, only N⁶-dAdo adducts were found in vivo, implying possible intervention by DNA repair mechanisms. Analogous to previous studies, levels of N⁶-POB-dAdo and N⁶-PHB-dAdo varied both with tissue and treatment type. Despite the adduct levels being relatively modest compared to most other POB- and PHB-DNA adducts, they may play a biological role and could be used in future studies as NNK- and NNAL-specific DNA damage biomarkers.

Full text of DOI:10.1021/acs.chemrestox.8b00056

Subscriber access provided by CAL STATE UNIV SAN FRANCISCO
Analysis and Identification of 2'-Deoxyadenosine-derived Adducts
in Lung and Liver DNA of F-344 Rats Treated with the Tobacco-
Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and
Enantiomers of its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol
Erik S Carlson, Pramod Upadhyaya, Peter W. Villalta, Bin Ma, and Stephen S. Hecht
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00056 • Publication Date (Web): 13 Apr 2018
Downloaded from http://pubs.acs.org on April 13, 2018
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Analysis and Identification of 2'ꢀDeoxyadenosineꢀderived Adducts in Lung and Liver DNA of Fꢀ344 Rats
Treated with the TobaccoꢀSpecific Carcinogen 4ꢀ(Methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone and
Enantiomers of its Metabolite 4ꢀ(Methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanol
Erik S. Carlson1,2, Pramod Upadhyaya1, Peter W. Villalta1, Bin Ma1, and Stephen S. Hecht1*
1
Masonic Cancer Center, University of Minnesota, 2231 6th Street SE, 2ꢀ210 CCRB, Minneapolis, MN
55455, United States
2
Department of Pharmacology, University of Minnesota Medical School, 321 Church Street SE, 6ꢀ120
Jackson Hall, Minneapolis, MN 55455
*To whom correspondence should be addressed:
Masonic Cancer Center, University of Minnesota, 2231 6th Street SE ꢀ 2ꢀ148 CCRB, Minneapolis, MN 55455, USA. phone: (612)
624ꢀ7604 fax: (612) 624ꢀ3869 eꢀmail: hecht002@umn.edu
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Chemical Research in Toxicology
Abstract:
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4ꢀ(Methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (NNK) and its metabolite 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀ
butanol (NNAL) are carcinogenic in animal models and are believed to play an important role in human lung
carcinogenesis in cigarette smokers. Cytochrome P450ꢀmediated metabolism of these tobaccoꢀspecific nitrosamines
produces reactive species that alkylate DNA in the form of pyridyloxobutyl (POB)ꢀ or pyridylhydroxybutyl (PHB)ꢀDNA
adducts. Understanding the formation mechanism and overall levels of these adducts can potentially enhance cancer
prevention methods through the identification of particularly susceptible smokers. Previous studies have identified and
measured a panel of POBꢀ and PHBꢀDNA base adducts of dGuo, dCyd, and Thd; however, dAdo adducts have yet to be
determined. In this study, we complete this DNA adduct panel by identifying and quantifying levels of NNKꢀ and NNALꢀ
derived dAdo adducts in vitro and in vivo. To accomplish this, we synthesized standards for expected dAdoꢀderived DNA
adducts and used isotopeꢀdilution LCꢀESI+ꢀMS/MS to identify POB adducts formed in vitro from the reaction of 4ꢀ
(acetoxymethylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (NNKOAc) with calf thymus DNA. Adduct levels were then
quantified in lung and liver DNA of rats chronically treated with NNK or NNAL for 50 weeks using similar LCꢀMS
detection methods. The in vitro studies identified N6ꢀPOBꢀdAdo and N1ꢀPOBꢀdIno as products of the reaction of
NNKOAc with DNA, which supports our proposed mechanism of formation. Though both N6ꢀdAdo and N1ꢀdIno adducts
were found in vitro, only N6ꢀdAdo adducts were found in vivo, implying possible intervention by DNA repair
mechanisms. Analogous to previous studies, levels of N6ꢀPOBꢀdAdo and N6ꢀPHBꢀdAdo varied both with tissue and
treatment type. Despite the adduct levels being relatively modest compared to most other POBꢀ and PHBꢀDNA adducts,
they may play a biological role and could be used in future studies as NNKꢀ and NNALꢀspecific DNA damage
biomarkers.
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Introduction:
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Tobacco use remains an important global health crisis by claiming nearly 6 million lives per year, a figure which is
expected to rise to 8 million annually by 2030.1 In the United States alone, 15% of the adult population are cigarette
smokers,2 resulting in ~480,000 preventable deaths annually.3 Of these, 160,000 are due to cancer which accounts for 30%
of all annual U.S. cancer deaths.4 Though tobacco use causes a variety of cancers, lung cancer is of particular concern as
~70ꢀ80% of all lung cancers are tobaccoꢀrelated,3,4 and each year, 1.5 million lung cancer deaths occur worldwide.
Understanding the mechanisms of tobaccoꢀinduced carcinogenesis can potentially lead to new approaches to cancer
prevention by identifying cigarette smokers particularly susceptible to lung cancer and other tobaccoꢀrelated cancers. One
important carcinogen in tobacco and tobacco smoke is 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (NNK, 2), a wellꢀ
established genotoxic agent evaluated, along with the related carcinogen N′ꢀnitrosonornicotine, as carcinogenic to humans
by the International Agency for Research on Cancer.5–7 NNK is found solely in tobacco products and readily causes
adenocarcinoma of the lung in rats, mice, hamsters, and ferrets regardless of the route of administration and at relatively
low doses.5 Additionally, epidemiological studies have correlated NNK metabolites in smokers’ urine to future lung
cancer onset.8–10
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An overview of NNK metabolism leading to DNA adduct formation is presented in Scheme 1. NNK is converted in
humans to both enantiomers of the carcinogenic metabolite 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanol (NNAL, 3) by
enzymatic carbonyl reduction.5,11 Studies suggest that this process may be partially reversible as (S)ꢀNNAL can be
enzymatically oxidized to NNK in vivo while (R)ꢀNNAL commonly remains reduced.12 Both enantiomers are Oꢀ or Nꢀ
glucuronidated and excreted in the urine.13 These and unmodified NNAL serve as useful biomarkers and were the basis of
the epidemiology studies noted above.8–10
Both NNK and NNAL are metabolized by P450s, activating them to DNAꢀalkylating agents.5 Initial hydroxylation of
the terminal methyl group of NNK or NNAL by either P450 2A6 or P450 2A1314 produces the unstable αꢀhydroxy
species 4 or 5. A small fraction of these may be converted further to nitrosamides,15 but most will rapidly decompose to
diazohydroxides 6 and 7, before losing H2O and forming highly reactive diazonium ions 8 and 9. These intermediates
hydrolyze to form metabolites 10 and 11, which are excreted in the urine after further enzymatic processing. However, 8
and 9 can also react with DNA to form a variety of products referred to as pyridyloxobutylꢀ (POB) or
pyridylhydroxybutylꢀ (PHB) DNA adducts, respectively. The POBꢀDNA adducts can also be formed in vitro from
NNKOAc, a precursor to 4 that does not require P450 metabolism. Analogously, the αꢀmethylene group can be
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hydroxylated to ultimately form methyl DNA adducts. DNA adducts left unrepaired can cause mutations and initiate
tumor development.5,6 To date, only four POBꢀ and three PHBꢀ base adducts have been detected and quantified in animal
models (Figure 1).12,16–18 In addition to these base adducts, a large family of DNA phosphate adducts have recently been
uncovered and quantified in vivo (Figure 1).19,20
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Notably missing from this panel of adducts are those formed with 2′ꢀdeoxyadenosine (dAdo). Therefore, the goal of
this study was to determine the structure and levels of dAdoꢀderived POBꢀ and PHBꢀadducts in NNKꢀ and NNALꢀtreated
rats. We hypothesized that initial alkylation of dAdo would occur at the N1ꢀposition (Scheme 2). This results in an
unstable cationic intermediate (19), which can deprotonate and undergo spontaneous Dimroth rearrangement to give N6ꢀ
dAdo adducts 20 and 21.21–23 Alternatively, deamination via an additionꢀelimination mechanism can occur yielding N1ꢀ
dIno adducts 22 and 23.23–26 In support of this general mechanism, N6ꢀdAdo and N1ꢀdIno type adducts have been found
after in vitro treatment of DNA with styrene oxide and butadiene monoxide.27,28 Along with these adduct types, previous
studies have identified N3ꢀ and N7ꢀdAdo adducts, but these are known to be unstable and generate abasic sites,29,30 thus
were not considered in this study. N1ꢀdIno adducts are thought to be potentially important as they can cause A:G
mutations through Hoogstein base pairing,31 while N6ꢀdAdo adducts may interfere with base pairing due to their steric
bulk. Regardless of their potential mutagenicity, these adducts also may serve as DNA damage biomarkers.32 Therefore, to
meet the goals of this study, we synthesized standards for all four proposed dAdoꢀderived adducts, detected POB adduct
formation in vitro, and quantified dAdo adduct formation in the livers and lungs of rats treated chronically with NNK and
NNAL.
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Experimental Procedures:
Caution: NNK and NNAL are carcinogenic in animal models. NNKOAc, an activated form of NNK, is mutagenic and is
presumed to be carcinogenic. Handle these compounds in a wellꢀventilated fume hood while wearing proper personal
protective equipment.
Chemicals and Enzymes:
NNK and NNKOAc were purchased from Toronto Research Chemicals. (S)ꢀNNAL, (R)ꢀNNAL, N6ꢀPHBꢀdAdo, 2ꢀ(3ꢀ
pyridyl)ꢀ1,3ꢀdithiane, tertꢀbutyl 3ꢀ(2ꢀ(3ꢀpyridyl)ꢀ1,3ꢀdithianyl)ꢀ1ꢀpropylcarbamate, and [pyridineꢀD4]tertꢀbutyl 3ꢀ(2ꢀ(3ꢀ
pyridyl)ꢀ1,3ꢀdithianyl)ꢀ1ꢀpropylcarbamate were synthesized as previously described.15,33,34 [15N5]2′ꢀdeoxyadenosine
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(>99% 15N5 incorporation) was obtained from Cambridge Isotope Laboratories (Tewksbury, MA). 6ꢀChloropurineꢀ2′ꢀ
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deoxyriboside was obtained from Carbosynth (Compton, United Kingdom). Reagents for DNA isolation were purchased
from Qiagen (Hilden, Germany). Calf thymus DNA, phosphodiesterase II, and micrococcal nuclease were obtained from
Worthington Biochemical Co. (Lakewood, NJ). Alkaline phosphatase was procured from Roche Diagnostics GmbH
(Mannheim, Germany). Porcine liver esterase and all other chemicals and solvents were obtained from either Sigma
Aldrich (Milwaukee, WI) or Thermo Scientific (Waltham, MA) in reagent grade or higher and used without further
purification.
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General Synthetic Procedures:
NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are reported as parts per million
(ppm). Residual solvent peaks were used as an internal reference for 1HꢀNMR (7.26 ppm CDCl3; 2.50 ppm D6ꢀDMSO)
and 13CꢀNMR (77.2 ppm CDCl3; 39.5 ppm D6ꢀDMSO). Peak splitting was abbreviated as follows: s = singlet, d = doublet,
t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, ddd = doublet of doublet of doublets, bs = broad
singlet, and m = multiplet. High resolution mass spectrometry (HRMS) of selected compounds was performed on an LTQ
Orbitrap Velos (Thermo Scientific, Waltham, MA) and reported as m/z. Thinꢀlayer chromatography (TLC) utilized
Polygram preꢀcoated silica gel TLC plate (40 x 80 mm, 0.2 mm thick) with 254 nm fluorescent indicator. TLC plates were
visualized with permanganate stain when necessary, otherwise UV lamp irradiation sufficed. Flash chromatography was
performed on SiliCycle 60 (70ꢀ150) mesh silica gel. Reactions were performed with ovenꢀdried glassware and under an
atmosphere of N2 unless specified otherwise.
[15N5]6ꢀNꢀ(4ꢀ(3ꢀpyridyl)ꢀ4ꢀhydroxyꢀ1ꢀbutyl)ꢀ2′ꢀdeoxyadenosine ([15N5]N6ꢀPHBꢀdAdo, 21):
This was prepared as previously described34 for N6ꢀPHBꢀdAdo except using [15N5]2′ꢀdeoxyadenosine as starting
material.
1HꢀNMR (500 MHz; DMSOꢀd6): δ 8.51 (d, J = 10.4 Hz, 1H, 2ꢀPyr), 8.42 (d, J = 4.7 Hz, 1H, 6ꢀPyr), 8.33ꢀ8.29 (m, 1H,
C8), 8.18 (s, 1H, C2), 7.87 (bs, 1H, NH), 7.71ꢀ7.69 (m, 1H, 4ꢀPyr), 7.32 (dd, J = 7.8, 4.8 Hz, 1H, 5ꢀPyr), 6.34 (t, J = 7.0
Hz, 1H, C1'), 5.31 (m, 2H, 3'ꢀOH/CHOH), 5.24 (s, 1H, 5'ꢀOH), 4.62 (m, 1H, CH), 4.41ꢀ4.40 (m, 1H, C3'), 3.88 (d, J = 2.2
Hz, 1H, C4'), 3.62 (m, 1H, C5'), 3.50 (m, 3H, C5'/NHCH2), 2.72 (dt, J = 12.0, 5.5 Hz, 1H, C2'), 2.25 (dd, J = 12.4, 4.4 Hz,
1H, C2'), 1.69ꢀ1.54 (m, 4H, CH2CH2).
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13CꢀNMR: (126 MHz; DMSOꢀd6): δ 152.6 (C2), 148.4 (6ꢀPyr), 148.1 (2ꢀPyr), 139.5 (C8), 133.9 (4ꢀPyr), 123.5 (5ꢀ
Pyr), 88.5 (C4'), 84.3 (C1'), 71.4 (C3'), 70.4 (CH), 62.4 (C5'), 39.9 (C2'), 39.8 (NCH2), 36.8 (CHCH2)), 25.9
(NCH2CH2). 3ꢀPyr, C4, C5, and C6 not observed.
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HRMS: calc’d: 406.17835; found: 406.17756
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3ꢀ(2ꢀ(3ꢀPyridyl)ꢀ1,3ꢀdithianyl)ꢀ1ꢀpropylamine (26):
A solution of 25 (87.6 mg, 0.247 mmol) in CH2Cl2 (3 mL) was treated with trifluoroacetic acid (TFA, 1,49 g, 13.1
mmol, 1 mL) and stirred at room temperature. After 3h, the solution was concentrated by rotary evaporation to remove
excess TFA. The resulting oil was dissolved in CH2Cl2 and washed with sat’d NaHCO3 solution and brine. The organic
layer was then dried over MgSO4, filtered, and concentrated in vacuo to give a yellow oil. Purification by column
chromatography (CH2Cl2/MeOH/Et3N 85:15:1) yielded the product as a light, yellow oil (41.4 mg, 66%).
1HꢀNMR (500 MHz; CDCl3): δ 9.11 (d, J = 1.8 Hz, 1H), 8.49ꢀ8.48 (m, 1H), 8.21 (d, J = 8.1 Hz, 1H), 7.31 (dd, J =
8.0, 4.8 Hz, 1H), 2.72ꢀ2.64 (m, 6H), 2.10ꢀ2.06 (m, 2H), 1.95 (dd, J = 8.9, 4.7 Hz, 2H), 1.53ꢀ1.49 (m, 2H). 13CꢀNMR (126
MHz; CDCl3): δ 150.5, 148.1, 137.6, 136.9, 123.3, 56.3, 42.1, 41.2, 27.5, 26.2, 24.9
6ꢀNꢀ(4ꢀ(3ꢀpyridyl)ꢀ4ꢀoxoꢀ1ꢀbutyl)ꢀ2'deoxyadenosine (N6ꢀPOBꢀdAdo, 20):
A solution of 26 (6.35 mg, 0.0250 mmol), 6ꢀchloropurineꢀ9ꢀ2'ꢀdeoxyriboside (10.4 mg, 0.0384 mmol), and iPr2EtN
(7.42 mg, 0.0574 mmol,10 ꢀL) in DMSO (300 ꢀL) was heated to 60 °C for 16 h. The compound was crudely isolated by
solidꢀphase extraction using a StrataꢀX 33 ꢀm polymeric reversed phase cartridge (30 mg/mL; Phenomenex, Torrance,
CA). The cartridge was preconditioned with 1 mL of MeOH, followed by 1 mL of H2O. After sample addition, the
cartridge was washed with 2 mL of H2O, 1 mL of 5% (v/v), 10%, and 50% MeOH, and eluted with 1 mL of 80% MeOH.
The eluent was evaporated to dryness in a Speedvac.
The isolated product was dissolved in 4:1 MeCN/H2O (1 mL) and added to a suspension of Nꢀchlorosuccinimide
(NCS, 11.9 mg, 0.0891 mmol) and AgNO3 (20.2 mg, 0.1188) in 4:1 MeCN/H2O (1 mL) at 0 °C. The reaction was stirred
at 0 °C for 30 min before quenching with sat’d Na2SO3, sat’d NaHCO3, and brine solutions in succession (1 mL each).
The product was isolated by HPLC using a 250 mm x 10 mm, 5 ꢀm, Luna Cꢀ18 column (Phenomenex) with H2O and
MeCN as mobile phases. The gradient was 30% to 45% MeCN over 5 min at 3 mL/min. The product eluted at 4.1 min.
The product was a light yellow solid (2.37 mg, 24% over two steps).
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1HꢀNMR (500 MHz; DMSOꢀd6): δ 9.09 (d, J = 1.1 Hz, 1H, 2ꢀPyr), 8.77 (dd, J = 4.7, 0.8 Hz, 1H, 6ꢀPyr), 8.32 (s, 1H,
Cꢀ8), 8.25 (d, J = 8.0 Hz, 1H, 4ꢀPyr), 8.12 (s, 1H, Cꢀ2), 7.94ꢀ7.93 (bs, 1H, NH), 7.54 (dd, J = 7.9, 4.8 Hz, 1H, 5ꢀPyr), 6.34
(t, J = 7.0 Hz, 1H, C1'), 5.30 (d, J = 3.9 Hz, 1H, 3'ꢀOH), 5.22 (t, J = 5.7 Hz, 1H, 5'ꢀOH), 4.41ꢀ4.40 (m, 1H, C3'), 3.88 (d, J
= 2.5 Hz, 1H, C4'), 3.64ꢀ3.50 (m, 4H, C5'+ NHCH2), 3.15 (t, J = 6.9 Hz, 2H, COCH2), 2.74ꢀ2.69 (m, 1H, C2'), 2.27ꢀ2.23
(m, 1H, C2'), 1.96 (quintet, J = 6.8 Hz, 2H, COCH2CH2). 13CꢀNMR (126 MHz; DMSOꢀd6): δ 199.5 (CO), 155.1 (C6),
153.7 (6ꢀPyr), 152.7 (C2), 149.6 (2ꢀPyr), 148.5 (C4), 139.8 (C8), 135.7 (4ꢀPyr), 132.4 (3ꢀPyr), 124.3 (5ꢀPyr), 120.1 (C5),
88.5 (C4'), 84.4 (C1'), 71.4 (C3'), 62.4 (C5'), 39.9 (C2'), 39.3 (NHCH2), 36.0 (COCH2), 21.6 (COCH2CH2).
HRMS: calc’d: 399.17753; found: 399.17742
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[PyridineꢀD4]3ꢀ(2ꢀ(3ꢀPyridyl)ꢀ1,3ꢀdithianyl)ꢀ1ꢀpropylamine:
This compound was prepared analogously to 26 except [pyridineꢀD4]25 (>98% D4ꢀincorporation) was used as starting
material. The product was a light yellow oil (7.67 mg, 35%).
1HꢀNMR (500 MHz; CDCl3): δ 2.71ꢀ2.64 (m, 4H), 2.61 (t, J = 7.0 Hz, 2H), 2.05 (dt, J = 7.9, 4.2 Hz, 2H), 1.98ꢀ1.93
(m, 2H), 1.47ꢀ1.41 (m, 2H).
13CꢀNMR (126 MHz; CDCl3): δ 56.5, 42.5, 41.8, 27.52, 27.42, 25.0
[PyridineꢀD4]6ꢀNꢀ(4ꢀ(3ꢀpyridyl)ꢀ4ꢀoxoꢀ1ꢀbutyl)2'ꢀdeoxyadenosine ([pyridineꢀD4]N6ꢀPOBꢀdAdo):
This compound was prepared analogously to N6ꢀPOBꢀdAdo except [pyridineꢀD4]26 (7.67mg, 0.0297 mmol) was used
as the starting material. The isolated product was a light yellow solid (2.08 mg, 18% over two steps).
1HꢀNMR (500 MHz; DMSOꢀd6): δ 8.32 (s, 1H, C8), 8.11 (s, 1H, C2), 7.94 (bs, 1H, NH), 6.33 (dd, J = 7.7, 6.2 Hz,
1H, C1'), 5.33 (d, J = 0.3 Hz, 1H, 3'ꢀOH), 5.24 (m, 1H, 5'ꢀOH), 4.41ꢀ4.40 (m, 1H, C3'), 3.88 (q, J = 3.4 Hz, 1H, C4'),
3.62ꢀ3.50 (m, 4H, C5'/COCH2), 3.15 (dd, J = 10.6, 5.9 Hz, 2H, NHCH2), 2.71ꢀ2.69 (m, 1H, C2'), 2.25 (ddd, J = 13.1, 6.1,
2.8 Hz, 1H, C2'), 1.96 (t, J = 6.9 Hz, 2H, CH2).
13CꢀNMR (126 MHz; DMSOꢀd6): δ 199.6 (CO), 155.1 (C6), 152.7 (C2), 147.0 (C4), 139.7 (C8), 132.3 (3ꢀPyr), 120.4
(C5), 88.5 (C4'), 84.4 (C1'), 71.4 (C3'), 62.3 (C5'), 39.6 (C2'), 39.2 (NHCH2), 36.1 (COCH2), 23.8 (COCH2CH2).
HRMS: calc’d: 403.20264; found: 403.20173
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3',5'ꢀbisꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2'ꢀdeoxyinosine (29):
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2'ꢀDeoxyinosine (463 mg, 1.84 mmol) and imidazole (554.5 mg, 8.14 mmol) were suspended in anhydrous pyridine
(5 mL) and treated with tertꢀbutyldimethylsilyl chloride (TBSꢀCl, 585 mg, 3.88 mmol). The mixture was stirred at room
temperature. After 4 h, the mixture became completely homogenous, and the reaction was quenched with H2O (10 mL).
The solution was extracted twice with CH2Cl2 (15 mL). The pooled organics were dried over MgSO4, filtered, and
concentrated. The resulting product was evaporated from toluene (5 mL) multiple times to remove residual pyridine.
NMR indicated that purity was sufficient to carry forward without further purification.
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1HꢀNMR (500 MHz; CDCl3): δ 12.61 (s, 1H), 8.19 (s, 1H), 8.08 (s, 1H), 6.40 (t, J = 6.4 Hz, 1H), 4.60 (dt, J = 5.6, 3.6
Hz, 1H), 4.01 (q, J = 3.5 Hz, 1H), 3.85ꢀ3.75 (m, 2H), 2.58ꢀ2.42 (m, 2H), 0.90 (d, J = 2.6 Hz, 18H), 0.10ꢀ0.08 (m, 12H).
13C NMR (126 MHz; CDCl3): δ 158.7, 148.3, 144.6, 138.4, 125.0, 88.0, 84.5, 71.7, 62.7, 41.6, 26.0, 25.7, 18.4, 18.0, ꢀ
4.67, ꢀ5.40.
1ꢀNꢀ(2,4ꢀdinitrophenyl)ꢀ3',5'ꢀbisꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2'ꢀdeoxyinosine (30):
A mixture of 29 (1384 mg, 2.14 mmol), K2CO3 (608.6 mg, 4.40 mmol), and 1ꢀchloroꢀ2,4ꢀdinitrobenzene (499.5 mg,
2.22 mmol) in DMF (10 mL) was heated to 80 °C for 2.5 h. The bright red solution turned orange during the reaction.
After 2.5 h, the reaction mixture was cooled to room temperature and diluted with EtOAc (10 mL). The mixture was
filtered over Celite and concentrated to a thick, red oil. Purification by column chromatography (EtOAc/hexanes 1:2
→1:1) yielded pure product as a yellow solid (1.26 g, 91% over 2 steps).
1HꢀNMR (500 MHz; CDCl3): δ 9.05 (d, J = 1.2 Hz, 1H), 8.68ꢀ8.66 (m, 1H), 8.21 (d, J = 10.3 Hz, 1H), 8.00 (s, 1H),
7.72ꢀ7.68 (m, 1H), 6.47ꢀ6.41 (m, 1H), 4.64ꢀ4.61 (m, 1H), 4.06ꢀ4.04 (m, 1H), 3.89ꢀ3.79 (m, 2H), 2.60ꢀ2.45 (m, 2H), 0.93
(s, 18H), 0.13ꢀ0.11 (m, 12H). 13CꢀNMR (126 MHz; CDCl3): δ 155.2, 148.2, 147.1, 146.50, 146.48, 144.72, 144.61, 139.2,
138.9, 135.9, 131.96, 131.83, 128.75, 128.70, 124.37, 124.17, 121.43, 121.38, 88.32, 88.18, 84.64, 84.44, 71.87, 71.71,
62.77, 62.63, 42.1, 41.8, 26.0, 25.7, 18.4, 18.0, ꢀ4.63, ꢀ4.66, ꢀ5.37, ꢀ5.48
1ꢀNꢀ(3ꢀhydroxypropyl)ꢀ3',5'ꢀbisꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2'ꢀdeoxyinosine (31):
A solution of 30 (701.2 mg, 1.08 mmol) was dissolved in DMF (10 mL) and treated with 3ꢀaminoꢀ1ꢀpropanol (810.2
mg, 10.8 mmol, 820 ꢀL). The yellow solution immediately became red. The solution was brought to 80 °C for 2 h, then
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back to room temperature. It was concentrated to a dark red oil. Purification by column chromatography (EtOAc/hexanes
2:1 → 1:0) yielded pure product as a yellow oil that solidified upon refrigeration (522.9 mg, 90%).
1HꢀNMR (500 MHz; CDCl3): δ 8.08 (s, 1H), 8.03 (s, 1H), 6.35 (t, J = 6.4 Hz, 1H), 4.58 (dt, J = 5.7, 3.1 Hz, 1H), 4.24
(t, J = 6.4 Hz, 2H), 3.99 (d, J = 3.4 Hz, 1H), 3.82ꢀ3.73 (m, 2H), 3.59 (t, J = 5.5 Hz, 2H), 2.52 (dt, J = 13.0, 6.4 Hz, 1H),
2.40 (ddd, J = 13.0, 5.9, 4.0 Hz, 1H), 1.97 (quintet, J = 5.9 Hz, 2H), 0.89 (d, J = 3.9 Hz, 18H), 0.09ꢀ0.06 (m, 12H).
13CꢀNMR (126 MHz; CDCl3): δ 157.4, 147.29, 147.18, 138.5, 124.6, 88.0, 84.2, 71.8, 62.7, 57.9, 43.1, 41.5, 32.6,
25.9, 18.4, 18.0, ꢀ4.7, ꢀ5.4
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1ꢀNꢀ(3ꢀiodopropyl)ꢀ3',5'ꢀbisꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2'ꢀdeoxyinosine (32):
Imidazole (69.0 mg, 1.01 mmol) and triphenylphosphine (222.7 mg, 0.849 mmol) were dissolved in CH2Cl2 (7 mL)
and brought to 0 °C. To this was added I2 (218.8 mg, 0.862 mmol). After 15 min, the color of the mixture changed from
yellow to dark orange. A CH2Cl2ꢀdiluted solution of 31 (415.9 mg, 0.772 mmol, 7 mL) was then added. After 10 min, the
reaction was warmed to room temperature and stirred for 2 h. Then the mixture was filtered over Celite and washed with
sat’d Na2S2O3 solution. The organic layer was separated and further washed with brine, dried over MgSO4, filtered, and
concentrated to a yellow solid. Purification by column chromatography (EtOAc/hexanes 3:1) yielded pure product as a
yellow oil (461.4 mg, 95%).
1HꢀNMR (500 MHz; CDCl3): δ 8.26 (s, 1H), 8.08 (s, 1H), 6.39 (t, J = 6.4 Hz, 1H), 4.60 (dt, J = 5.9, 3.2 Hz, 1H), 4.19
(t, J = 6.6 Hz, 2H), 4.03 (q, J = 3.5 Hz, 1H), 3.85ꢀ3.76 (m, 2H), 3.17 (t, J = 6.4 Hz, 2H), 2.59ꢀ2.43 (m, 2H), 2.33 (quintet,
J = 6.5 Hz, 2H), 0.91 (d, J = 4.7 Hz, 18H), 0.11ꢀ0.08 (m, 12H). 13CꢀNMR (126 MHz; CDCl3): δ 155.5, 147.5, 146.9,
138.3, 123.8, 88.3, 84.7, 71.8, 62.7, 47.5, 41.6, 31.9, 26.0, 25.7, 18.4, 18.0, 2.3, ꢀ4.65, ꢀ4.78, ꢀ5.35, ꢀ5.45.
1ꢀNꢀ(3ꢀ(2ꢀ(3ꢀPyridyl)ꢀ1,3ꢀdithianyl)propyl)ꢀ3',5'ꢀbisꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2'ꢀdeoxyinosine (34):
A solution of 3315 (78.1 mg, 0.396 mmol) and tetramethylethylenediamine (TMEDA, 46.5 mg, 0.400 mmol, 60 ꢀL) in
anhydrous THF (4 mL) was cooled to ꢀ78 °C and treated with nꢀBuLi in hexanes dropwise (0.480 mmol, 300 ꢀL). The
resulting red solution was stirred for 10 min. A THFꢀdiluted solution of 32 (228.5 mg, 0.352 mmol, 3 mL) was then added
dropwise. The mixture was stirred for 4 h and the color slowly transitioned from red to yellow. The reaction was
quenched with H2O (5 mL) and warmed to room temperature. The mixture was diluted with EtOAc and washed with
brine. The aqueous phase was extracted twice with EtOAc. The pooled organics were dried over MgSO4, filtered, and
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concentrated to an orange oil. Purification by column chromatography (100% EtOAc) yielded the product as a yellow
solid (179.4 mg, 71%).
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1HꢀNMR (500 MHz; CDCl3): δ 9.12 (d, J = 1.9 Hz, 1H), 8.53 (d, J = 3.9 Hz, 1H), 8.30 (m, 1H), 8.05 (s, 1H), 7.85 (s,
1H), 7.42 (dd, J = 8.0, 4.9 Hz, 1H), 6.38ꢀ6.33 (m, 1H), 4.59ꢀ4.55 (m, 1H), 4.27 (s, 1H), 3.97 (m, 4H), 3.82ꢀ3.59 (m, 2H),
2.77ꢀ2.49 (m, 6H), 2.06 (dd, J = 9.8, 6.4 Hz, 2H), 1.98ꢀ1.76 (m, 4H), 0.78 (s, 18H), 0.07 (m, 12H).
13CꢀNMR (126 MHz; CDCl3): δ 156.5, 149.0, 147.2, 146.86, 146.69, 139.5, 138.6, 138.3, 124.9, 89.9, 84.8, 73.2,
63.5, 56.1, 46.2, 41.9, 41.7, 27.6, 26.1, 24.7, 18.6, 18.1, ꢀ4.53, ꢀ4.67, ꢀ5.35, ꢀ5.39
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1ꢀNꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀbutyl)ꢀ2'ꢀdeoxyinosine (N1ꢀPOBꢀdIno, 22):
NꢀChlorosuccinimide (NCS, 11.5 mg, 0.0861) and AgNO3 (15.5 mg, 0.0912) were suspended in 1:1 MeCN/H2O (400
ꢀL) and cooled to 0 °C. To this was added a solution of 34 in MeCN (11.1 mg, 0.0155 mmol, 600 ꢀL). The reaction
mixture was stirred at 0 °C for 30 min before quenching with sat’d Na2SO3, sat’d NaHCO3, and brine solutions in
succession (1 mL each). The mixture was diluted with CH2Cl2 (5 mL) and filtered. The organic layer was collected and
the aqueous phase further extracted once with CH2Cl2. The pooled organics were dried over MgSO4, filtered, and
concentrated to a yellow oil. The crude product was then transferred and concentrated into a 2ꢀmL polypropylene tube and
reconstituted in THF (200 ꢀL). To this was added 5 ꢀL of a K2HPO4ꢀbuffered solution of tetraꢀnꢀbutylammonium fluoride
(TBAF, 3.4M, 25mM 2:1 K2PO4, pH 7.1/THF). The solution immediately turned orange and was shaken for 24 h. The
solvent was evaporated to give an orange solid, which was reconstituted in H2O (2 mL) and purified by HPLC using a 250
mm x 10 mm, 5 ꢀm, Luna Cꢀ18 column (Phenomenex) with H2O and MeCN as mobile phases. The gradient was 30% to
45% MeCN over 5 min at 3 mL/min. The product eluted at 10.7 min. The product was a white solid (2.37 mg, 24% over
two steps).
1HꢀNMR (500 MHz; DMSOꢀd6): δ 9.09 (d, J = 1.7 Hz, 1H, 2ꢀPyr), 8.78 (dd, J = 4.8, 1.6 Hz, 1H, 6ꢀPyr), 8.42 (s, 1H,
C2), 8.31 (s, 1H, C8), 8.25 (dt, J = 8.0, 1.9 Hz, 1H, 4ꢀPyr), 7.56 (dd, J = 7.9, 4.8 Hz, 1H, 5ꢀPyr), 6.30 (t, J = 6.9 Hz, 1H,
C1'), 5.32 (d, J = 3.7 Hz, 1H, 3'ꢀOH), 4.97 (t, J = 5.5 Hz, 1H, 5'ꢀOH), 4.39ꢀ4.37 (m, 1H, C3'), 4.09 (t, J = 7.0 Hz, 2H,
NCH2), 3.86 (q, J = 3.7 Hz, 1H, C4'), 3.61ꢀ3.49 (m, 2H, C5'), 3.16 (t, J = 7.0 Hz, 2H, COCH2), 2.62 (ddd, J = 13.3, 7.2,
6.1 Hz, 1H, C2'), 2.31ꢀ2.27 (m, 1H, C2'), 2.05 (t, J = 7.0 Hz, 2H, COCH2CH2).
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13CꢀNMR (126 MHz; DMSOꢀd6): δ 198.6 (CO), 156.1 (C6), 153.3 (6ꢀPyr), 149.1 (2ꢀPyr), 148.4 (C2), 147.1 (C4),
139.3 (C8), 135.3 (4ꢀPyr), 131.8 (3ꢀPyr), 123.8 (5ꢀPyr), 123.4 (C5), 88.6 (C4'), 83.6 (C1'), 70.6 (C3'), 61.5 (C5'), 45.2
(NCH2), 40.4 (C2'), 35.2 (COCH2), 23.5 (COCH2CH2)
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1ꢀNꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀbutyl)ꢀ 3',5'ꢀbisꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2'ꢀdeoxyinosine (35):
NꢀChlorosuccinimide (NCS, 12.9 mg, 0.0966) and AgNO3 (20.6 mg, 0.121) were suspended in 1:1 MeCN/H2O (200
ꢀL) and cooled to 0 °C. To this was added a solution of 34 in 1:1 MeCN:H2O (15.6 mg, 0.0217 mmol, 800 ꢀL). The
reaction mixture was stirred at 0 °C for 30 min before quenching with sat’d Na2SO3, sat’d NaHCO3, and brine solutions in
succession (1 mL each). The mixture was diluted with CH2Cl2 (5 mL) and filtered. The organic layer was collected and
the aqueous phase extracted once with CH2Cl2. The pooled organics were dried over MgSO4, filtered, and concentrated to
a yellow oil. The crude product was then dissolved in MeOH (800 ꢀL) and treated with NaBH4 (7.0 mg, 0.185 mmol) at
room temperature. After 1 h, the reaction mixture was quenched with sat’d NH4Cl, diluted with CH2Cl2 and washed with
sat’d NaHCO3. The aqueous phase was further extracted three times with CH2Cl2. The pooled organics were dried over
MgSO4, filtered, and concentrated to a white solid (6.15 mg). The crude material was carried forward without further
purification.
1ꢀNꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀbutyl)ꢀ2'ꢀdeoxyinosine (N1ꢀPHBꢀdIno, 23):
A solution of 35 (3.4 mg, 0.00540 mmol) in THF (450 ꢀL) was treated with TBAF in THF (0.0150 mmol, 15 ꢀL) at
room temperature. After 1 h, the reaction mixture was diluted with H2O and filtered through a 4 mm Acrodisc syringe
filter (Sigma Aldrich). The resulting solution was purified by HPLC using a 250 mm x 10 mm, 5 ꢀm, Luna Cꢀ18 column
(Phenomenex) with H2O and MeCN as mobile phases. The gradient was 30% to 45% MeCN over 5 min at 3 mL/min. The
product eluted at 7.1 min. The product was a white solid (1.61 mg, 34% over 3 steps).
1HꢀNMR (500 MHz; DMSOꢀd6): δ 8.51ꢀ8.51 (m, 1H, 2ꢀPyr), 8.43 (dd, J = 4.7, 1.5 Hz, 1H, 6ꢀPyr), 8.40 (s, 1H, C2),
8.33 (s, 1H, C8), 7.70 (dd, J = 7.8, 1.6 Hz, 1H, 4ꢀPyr), 7.33 (dd, J = 7.7, 4.7 Hz, 1H, 5ꢀPyr), 6.29 (dd, J = 7.7, 2.8 Hz, 1H,
C1'), 5.43 (m, 2H, 3'ꢀOH/5'ꢀOH), 4.85 (s, 1H, OH), 4.63ꢀ4.61 (m, 1H, CH), 4.32ꢀ4.29 (m, 1H, C3'), 4.14ꢀ4.11 (m, 1H,
C4'), 4.03ꢀ4.00 (m, 2H, NCH2), 3.48ꢀ3.41 (m, 2H, C5'), 2.76ꢀ2.70 (m, 1H, C2'), 2.33ꢀ2.29 (m, 1H, C2'), 1.79ꢀ1.56 (m, 4H,
CH2CH2).
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13CꢀNMR (126 MHz; DMSOꢀd6): δ 156.4 (C6), 148.8 (C2), 148.5 (6ꢀPyr), 148.0 (2ꢀPyr), 146.8 (C4), 139.8 (C8),
136.5 (3ꢀPyr), 133.8 (4ꢀPyr), 125.5 (C5), 123.7 (5ꢀPyr), 89.5 (C4'), 84.1 (C1'), 71.0 (C3'), 70.2 (CH), 62.0 (C5'), 46.1
(NCH2), 36.3 (CHCH2), 26.1 (CHCH2CH2).
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In vitro detection of POBꢀDNA adducts using NNKOAc:
Calf thymus DNA (0.99 mg) was dissolved in 1.0 mL of 25 mM phosphate buffer, pH 7.4. To this was added porcine
liver esterase (1.33 mg, 24U) and NNKOAc (1 mg, 3.7 ꢀmol in 20 ꢀL of MeOH) and the mixture was placed in a 37 °C
water bath with shaking for 24 h. Then, the solution was diluted with 1 mL of H2O and extracted twice with 2 mL of
CHCl3/isoamyl alcohol (24:1). The DNA was precipitated by addition of 0.4 mL of 5M NaCl and 2 mL of iceꢀcold
isopropyl alcohol. The DNA was further washed with 1 mL of 70% EtOH, followed by 1 mL of 100% EtOH and dried
under a stream of N2. DNA was stored at ꢀ20 °C.
Isolated DNA was dissolved in 0.5 mL of 10 mM sodium succinate, 5 mM CaCl2 buffer and incubated at 37 °C with
micrococcal nuclease (30 U) and phosphodiesterase II (0.5 U) for 5 h, followed by 37 °C incubation with alkaline
phosphatase (150 U) for 16 h. [PyridineꢀD4]N6ꢀPOBꢀdA (100 fmol) was added as an internal standard. Samples were
placed in 0.45 ꢀm Nylon SpinX centrifugal tube filters and centrifuged at 12,000 g for 10 min. A 20 ꢀL aliquot was
removed from each sample and diluted as necessary for dGuo quantiation by HPLC.18,35 The remaining sample was
purified by solidꢀphase extraction using a StrataꢀX 33 ꢀm polymeric reversed phase cartridge (30 mg/mL; Phenomenex,
Torrance, CA). The cartridge was preconditioned with 1 mL of MeOH, followed by 1 mL of H2O. After sample addition,
the cartridge was washed with 2 mL of H2O and 1 mL of 10% (v/v) MeOH, and eluted with 1 mL of 100% MeOH. The
eluent was evaporated to dryness in a Speedvac. The residue was dissolved in 25 ꢀL of H2O and analyzed by liquid
chromatographyꢀpositive electrospray ionizationꢀtandem mass spectrometry (LCꢀESI+ꢀMS/MS).
A previously reported LCꢀESI+ꢀMS/MS method was performed with modifications.18 Briefly, a 0.5 × 150 mm Zorbax
SBꢀC18, 5 ꢁm column (Agilent, Santa Clara, CA) was eluted with a multiꢀstep gradient and flow rate of 10 ꢁL/min. After
a 5ꢀmin hold at 5% B, the eluent was brought to 65% B over 25 min, followed by a wash at 85% B and reꢀequilibration,
where solvent A was 15 mM NH4OAc and solvent B was methanol. MS was performed on a TSQ Vantage triple
quadrupole mass analyzer (Thermo Scientific). The selected reaction monitoring (SRM) transitions were m/z 400.1 → m/z
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284.1 for N1ꢀPOBꢀdIno, m/z 399.1 → m/z 265.1 for N6ꢀPOBꢀdAdo, and m/z 403.1 → m/z 269.1 for [pyridineꢀD4]N6ꢀPOBꢀ
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In vivo detection and quantitation of POBꢀ and PHBꢀDNA adducts in rat liver and lung tissues:
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The lung and liver tissues were obtained from a previous study in which male Fꢀ344 rats were treated with 5 ppm of
NNK, (S)ꢀNNAL, or (R)ꢀNNAL in drinking water for 50 weeks.18,35 Control rats were given unmodified tap water. DNA
was isolated from these tissues (n = 3) using a modified Qiagen protocol that has been applied in previous studies.17,35,36
Isolated DNA was stored at ꢀ20 °C until further analysis.
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Isolated DNA samples were hydrolyzed and spiked with [pyridineꢀD4]N6ꢀPOBꢀdAdo and [15N5]N6ꢀPHBꢀdAdo as
described above for our in vitro experiment, then analyzed for adducts as described previously.17,18,36,37 LCꢀESI+ꢀMS/MS
conditions were the same as described above for our in vitro experiment except SRM transitions were added for N6ꢀPHBꢀ
dAdo (m/z 401.1 → m/z 132.1), N1ꢀPHBꢀdIno (m/z 402.1 → m/z 286.1), and [15N5]N6ꢀPHBꢀdAdo (m/z 406.1 → m/z 132.1)
at collision energies of 26, 10, 26 eV, respectively. Corresponding fragmentation patterns are shown in Figure S2.
Samples were further analyzed by an optimized liquid chromatographyꢀpositive nanoelectrospray ionizationꢀhigh
resolution tandem mass spectrometry (LCꢀNSI+ꢀHRMS/MS) method. A New Objective (Woburn, MA) emitter (75 ꢀm
ID, 10 ꢀm orifice) was handꢀpacked with Luna (Phenomenex) C18, 5 ꢀm stationary phase to create a 200 mm capillary
column and a multistep gradient was used for chromatographic separation. With 5 mM NH4OAc and MeCN as solvents A
and B, respectively, the sample was loaded onto the column by running 2% B at 1 ꢀL/min for 5 min. Then the flow rate
was decreased to 0.3 ꢀL/min over 1 min, the injection valve was removed from the flow path, and a linear gradient was
started that ramped from 2% B to 40% B over 20 min. This was followed by an increase to 98% B over 1 min, in which
the column was washed for 2 min at 1 ꢀL/min before a 5 min reꢀequilibration at 2% B. All dAdo and dIno adducts were
monitored by MS2 fragmentation on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Waltham, MA).
The spray voltage was 2.2 kV, the capillary temperature was 300 °C, and the SꢀLens RF level was 60%. Each parent ion
was isolated (1.5 amu isolation width) and fragmented by higherꢀenergy collision dissociation (HCD) at a normalized
energy of 22% with a resolution of 60,000 and mass range of m/z 100 – 425. Accurate product ion masses from
characteristic transitions for N1ꢀPOBꢀdIno (m/z 400 → 284.1140, 148.0757), N1ꢀPHBꢀdIno (m/z 402 → 286.1296,
268.1191), N6ꢀPOBꢀdAdo (m/z 399 → 283.1300, 265.1194), N6ꢀPHBꢀdAdo (m/z 401 → 287.1550, 269.1445), [D4]N6ꢀ
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POBꢀdAdo (m/z 403 → 285.1456, 267.1350), and [15N5]N6ꢀPHBꢀdAdo (m/z 406 → 290.1307, 272.1202) were extracted at
a mass tolerance of 5 ppm (Figure S2).
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Quantitation of other DNA base adducts (12, 14, and 15) for comparison was performed as previously described using
similar DNA isolation and LCꢀESI+ꢀMS/MS or LCꢀNSI+ꢀHRMS/MS techniques.18,19,38
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Results:
Synthesis of DNA adduct standards
N6ꢀPHBꢀdAdo was previously synthesized by a 2ꢀstep procedure34 (Scheme 3A) which was applied to produce a 15N5ꢀ
labelled standard starting from [15N5]2′ꢀdeoxyadenosine.
Initial attempts to oxidize N6ꢀPHBꢀdAdo to N6ꢀPOBꢀdAdo were unsuccessful. Therefore, we devised a new route that
utilized previously prepared compound 25 as starting material15 (Scheme 3B). After Boc deprotection by TFA, primary
amine 26 was coupled to 6ꢀchloropurineꢀ2′ꢀdeoxyribose. The synthesis was completed by a final oxidative dithiane
deprotection39 and HPLC purification resulting in a 17% overall yield. A [pyridineꢀD4]ꢀlabeled standard was synthesized
analogously using [pyridineꢀD4]25, which was prepared in 2 steps15 from commercially available [pyridineꢀD4]3ꢀ
pyridylcarboxaldehyde.
The synthesis of N1ꢀPOBꢀdIno (22, Scheme 4A) began with protection of both hydroxyl groups of 2′ꢀdeoxyinosine
with tertꢀbutyldimethylsilyl chloride and N1ꢀarylation with 1ꢀchloroꢀ2,4ꢀdinitrobenzene to produce 30 as a 1:1 mixture of
atropisomers in excellent yield. Compound 30 was heated with 3ꢀaminoꢀ1ꢀpropanol to give 31 in good to excellent yield
through a transamidationꢀlike mechanism.40 This was converted into iodide 3241 and further coupled to dithiane 34 under
Seebach’s conditions42 with good yields for both steps. Lastly, the keto and hydroxyl groups were deprotected by Nꢀ
chlorosuccinimide oxidation and a pHꢀneutral, catalytic TBAF procedure,43 respectively, to produce N1ꢀPOBꢀdIno (22) in
modest yield over these two steps.
N1ꢀPHBꢀdIno (23, Scheme 4B) was obtained by reducing the ketone after deprotection of 34 and removing the TBS
groups with an unbuffered TBAF solution with moderate yield. All final DNA adduct structures were confirmed by oneꢀ
and twoꢀdimensional NMR experiments (1H, 13C, HSQC, HMBC) and highꢀresolution mass spectrometry.
In vitro detection of 2'ꢀdeoxyadenosineꢀderived POB adducts:
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Mixtures of calf thymus DNA, NNKOAc, and porcine liver esterase were incubated at 37 °C and pH 7.4 for 24 h.
After DNA hydrolysis and enrichment, samples were analyzed by LCꢀESI+ꢀMS/MS using [pyridineꢀD4]N6ꢀPOBꢀdAdo as
an internal standard. As shown in Figure 2, peaks corresponding to both N6ꢀPOBꢀdAdo and N1ꢀPOBꢀdIno were observed.
Based on relative quantitation, N1ꢀPOBꢀdIno was 25 times more abundant than N6ꢀPOBꢀdAdo. These peaks were not
detected in control incubations that either contained esterase alone or lacked NNKOAc. This indicates that both
deamination and Dimroth rearrangement occur under these conditions and implicates the N1ꢀposition as the initial site of
alkylation.
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In vivo detection and quantification of 2'ꢀdAdo adducts:
DNA was extracted from the livers and lungs of rats chronically treated with 5 ppm NNK, (S)ꢀNNAL, or (R)ꢀNNAL
in the drinking water for 50 weeks.18 Hydrolysis and analysis by LCꢀESI+ꢀMS/MS of these samples were performed as in
our in vitro experiment. Representative chromatograms for lung samples are shown in Figure 3. N6ꢀPOBꢀdAdo and N6ꢀ
PHBꢀdAdo were detected, while both N1ꢀPOBꢀdIno and N1ꢀPHBꢀdIno remained unobserved. Though a characteristic peak
at 20.9 min, possibly corresponding to N1ꢀPOBꢀdIno, was found in the m/z 400 → m/z 148 SRM channel, the
corresponding peak in the m/z 400 → m/z 284 SRM channel was not observed. The 20.9 min peak was detected in all
control samples indicating this signal is ubiquitous to the sample matrix. This same trend was also seen for N1ꢀPHBꢀdIno.
In the liver, levels of N6ꢀPOBꢀdAdo were higher in the NNK and (S)ꢀNNAL treatment groups (27ꢀ32 fmol/mg DNA
or 9ꢀ10.7 adducts/109 nts) as compared to the (R)ꢀNNAL group (4 fmol/mg DNA or 1.3 adduct/109 nts). The trend was
reversed for N6ꢀPHBꢀdAdo in that levels were lower in the NNK and (S)ꢀNNAL treatment groups (4ꢀ6 fmol/mg DNA or
1ꢀ2 adducts/109 nts) than in the (R)ꢀNNAL group (21ꢀ25 fmol/mg DNA or 7ꢀ8.3 adducts/109 nts) (Figure 4). Analysis of
rat lung DNA revealed the same trend, however, at higher overall levels. N6ꢀPOBꢀdAdo was relatively abundant in the
NNK and (S)ꢀNNAL groups at 99 fmol/mg DNA (33 adducts/109 nts) and 130 fmol/mg DNA (43.3 adducts/109 nts),
respectively, while N6ꢀPHBꢀdAdo was relatively high in the (R)ꢀNNAL group at 125 fmol/mg DNA (41.7 adducts/109
nts). Similar to the liver, N6ꢀPOBꢀdAdo was a minor product after (R)ꢀNNAL treatment (16 fmol/mg DNA or 5.3
adducts/109 nts) and N6ꢀPHBꢀdAdo was less abundant in both NNK and (S)ꢀNNAL treatment groups (35 fmol/mg DNA
or 11.7 adducts/109 nts). Control rats given unmodified tap water showed no adduct formation. To evaluate if the lack of
dIno adducts was due to insufficient sensitivity, these samples were reanalyzed by a LCꢀNSI+ꢀHRMS/MS method
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utilizing accurate mass detection. After extensive method optimization, the dIno adducts were still undetectable, while
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dAdo adducts were easily observed (Figure S1).
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Discussion:
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We describe the first characterization of dAdo base adducts formed in the metabolism of NNK by αꢀhydroxylation.
Synthesis of the standards N6ꢀPOBꢀdAdo, N6ꢀPHBꢀdAdo, N1ꢀPOBꢀdIno, and N1ꢀPHBꢀdIno as well as stable isotopeꢀ
labelled analogues allowed us to interrogate their presence and abundance in vitro and in vivo. Detection of both N6ꢀdAdo
and N1ꢀdIno type adducts in vitro, from reactions of NNKOAc with calf thymus DNA, supports our hypothesized
mechanism of formation involving either a spontaneous Dimroth rearrangement or deamination, respectively. Likewise,
quantitation of the N6ꢀdAdo adducts in liver and lung DNA of NNK and NNALꢀtreated rats supports the occurrence of
this mechanism in vivo.
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Levels of the dAdo adducts in liver and lung of rats treated chronically with NNK or enantiomers of NNAL in their
drinking water for 50 weeks are compared to those of some other characterized NNKꢀDNA adducts19 in Table 1. dAdo
adduct levels were relatively low in both tissues, exceeding only those of O6ꢀPOBꢀdGuo when measurable. Levels were
otherwise considerably lower than the 7ꢀGua and O2ꢀThd POBꢀ and PHBꢀDNA adducts. Levels of the dAdo adducts were
higher in lung than in liver of the NNK treated rats, consistent with the comparative tissue levels of the other DNA
adducts.18 This reflects extensive metabolic activation of NNK and NNAL in rat lung upon treatment with relatively low
doses of these carcinogens. Consistent with previous studies, levels of N6ꢀPOBꢀdAdo were higher in rats treated with
NNK or (S)ꢀNNAL than those found in rats treated with (R)ꢀNNAL. In comparison, N6ꢀPHBꢀdAdo amounts were highest
in lung and liver DNA of (R)ꢀNNALꢀtreated rats. These consistent findings presumably result from the facile conversion
of (S)ꢀNNAL to NNK in vivo and slow conversion of (R)ꢀNNAL to NNK as compared to direct metabolic activation by
P450s.12
In contrast to the results of our in vitro studies, in which NNKOAc was reacted with calf thymus DNA, we found no
evidence for the presence of either N1ꢀdIno adduct in lung and liver DNA of rats treated with NNK or enantiomers of
NNAL. This was in spite of the use of a highly sensitive and specific LCꢀNSI+ꢀHRMS/MS method. The accurate mass
detection feature of this method, when coupled with nanoꢀspray ionization, guarantees both high specificity and sensitivity
in detection of analytes. Even after rigorous application of this method, the dIno adducts remained undetected, perhaps
due to their efficient repair in vivo. Since other N1ꢀdIno adducts form Hoogstein base pairs and flip outside the DNA helix,
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it is possible that our POBꢀ and PHBꢀN1ꢀdIno adducts are prime targets for base excision repair or other DNA repair
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In our preliminary studies of the reaction of NNKOAc with calf thymus DNA, we observed an HPLC peak which
decreased relatively rapidly with time, as peaks corresponding to N6ꢀPOBꢀdAdo and N1ꢀPOBꢀdIno increased (data not
shown). We presume that this peak was N1ꢀPOBꢀdA. Ultimately, the concentration of N1ꢀPOBꢀdIno was about 25 times
greater than that of N6ꢀPOBꢀdAdo in these in vitro reactions. This preference is consistent with previous studies where
deamination dominated under neutral pH and when the N1ꢀalkyl group possessed a nucleophilic oxygen group.23–26 In the
case of styrene oxide, the latent alcohol was shown to facilitate the additionꢀelimination mechanism of deamination,24
however, it is unclear how these results translate to the POBꢀ and PHBꢀadducts. While Dimroth rearrangement is the most
plausible explanation for the presence of N6ꢀPOBꢀdAdo and N6ꢀPHBꢀdAdo, direct N6ꢀalkylation cannot be excluded as
there is some evidence for this in reactions of acrolein46 and 1,2,3,4ꢀdiepoxybutane47 with DNA.
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Synthesis of the adduct standards proceeded as expected with the exception of two major findings. First, the
transformation of 30 (Scheme 4) to N1ꢀalkyl structures showed a dependence on steric features. It was our original plan to
directly produce 34 by using 26 (Scheme 3 & 4) as the nucleophile, but this reaction gave little to no yield. We believe
that steric repulsion between the dithiane and TBSꢀgroups blocked the amino group from engaging the C2ꢀposition of the
nucleoside. Therefore, we simplified the attacking group to 3ꢀaminoꢀ1ꢀpropanol and obtained good yields of 31, which
was further elaborated to the final product. Next, we found TBSꢀdeprotection of 34 to be very sensitive to pH. After
dithiane removal, unbuffered TBAF removed not only the TBS groups, but also the entire POB moiety, perhaps due to the
basicity of fluoride. We surmise that N1ꢀPOB cleavage occurred via an enolateꢀring closure that released dIno and
presumably a cyclopropyl byproduct.48 When recently developed conditions using catalytic fluoride and neutral pH buffer
were applied,43 selective TBS cleavage was observed. In agreement with the enolateꢀring closure hypothesis, unbuffered
TBAF could be used to deprotect compound 35 with no cleavage of the PHB group.
Though adduct levels were relatively low in vivo, they may play a biological role. As discussed earlier, O6ꢀPOBꢀdGuo
is found at very low or undetectable levels in vivo (Table 1); however, this adduct is thought to contribute to
tumorigenesis as it is strongly mutagenic in a variety of cell models.44 Though efficient repair tempers this mutagenicity in
vivo, it may increase the persistence of other adducts such as O6ꢀMeꢀdGuo, since previous work identified GC to AT
transitions as a major NNKꢀinduced mutation.49 The same study also found moderate levels of AT to GC mutations,
perhaps due to the persistence of N6ꢀdAdo adducts. This is reasonable as the bulky N6ꢀlinkage could block base pairing
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with Thd. In studies with 1,3ꢀbutadiene, particular N6ꢀdAdo adducts were bypassed by several DNA polymerases and in
some cases caused mutations in vitro.50 However, in a followꢀup study,51 the same adducts were found to be far less
mutagenic, possibly due to baseꢀexcision repair. In studies with polycyclic aromatic hydrocarbons (PAHs), N6ꢀdAdo
adducts were highly mutagenic, and sometimes tumorigenic, depending on the PAH structure and location on the DNA
strand.52,53 When adducts significantly distorted the DNA helix, cells were found to halt during DNA replication, possibly
alluding to DNA polymerases stalling near the adduct site.52 Together, these findings seem to indicate that effective N6ꢀ
dAdo adducts require steric balance; adducts should be large enough to disrupt base pairing, but small enough to evade
detection by cellular machinery. It is currently unknown how POBꢀ and PHBꢀ adducts fit into this context and thus
requires future study. Along with possible biological effects, N6ꢀPOBꢀdAdo and N6ꢀPHBꢀdAdo have potential as
biomarkers. These products directly link to the DNA damaging properties of NNK and NNAL and could be used to study
differences between demographic groups.32 With analytical power continuing to increase, measurement of low levels of
DNA adducts in humans is starting to become an achievable feat.54
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In summary, this work has identified and quantified the first dAdoꢀderived DNA adducts of NNK and its enantiomers
NNAL formed in vitro and in vivo. We found general support for our proposed adduct formation mechanism by
quantifying both N1ꢀPOBꢀdIno and N6ꢀPOBꢀdAdo in vitro. Though N1ꢀdIno type adducts were the major ones formed in
vitro, they were undetectable in vivo. N6ꢀdAdo adduct levels were relatively modest in vivo when compared to most DNA
adducts of NNK and NNAL previously detected. This research allows and encourages future studies on the biological
properties of these adducts and their potential utility as specific DNA damage biomarkers.
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Funding Information:
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This study was supported by grant no. CAꢀ81301 from the U.S. National Cancer Institute. Salary support for P.W.V. was
provided by the U.S. National Cancer Institute [Grant R50ꢀCA211256]. The Analytical Biochemistry Shared Resource is
partially supported by National Cancer Institute Cancer Center Support Grant CAꢀ77598.
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Acknowledgements:
We thank Bob Carlson for editorial assistance, Xun Ming and Makenzie Pillsbury for mass spectrometry assistance in the
Analytical Biochemistry Shared Resource of the Masonic Cancer Center, and Dr. Adam T. Zarth for valuable discussions
and NMR assistance.
Supporting Information:
LCꢀNSI+ꢀHRMS/MS chromatograms from the analysis of POBꢀ and PHBꢀDNA adducts in the lungs of rats chronically
treated with NNK, (S)ꢀNNAL, and (R)ꢀNNAL, and fragmentation patterns for all N6ꢀdAdo and N1ꢀdIno adducts. This
material is available free of charge via the Internet at http://pubs.acs.org
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Abbreviation List:
1
7ꢀPHBꢀGua – N7ꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀguanine
7ꢀPOBꢀGua – N7ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀguanine
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3
4
5
6
7
8
Boc – tertꢀbutylcarbamate
B1p(POB)B2 – B1p(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)B2
B1p(PHB)sB2 – B1p(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)B2
B1p(PHB)bB2 – B1p(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ2ꢀbutyl)B2
DIPEA – N,N'ꢀdiisopropylethylamine
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DMF – N,N'ꢀdimethylformamide
DMSO – dimethylsulfoxide
HCD – Higherꢀenergy collisional dissociation
Imid – imidazole
LCꢀESI+ꢀMS/MS – liquid chromatographyꢀpositive electrospray ionizationꢀtandem mass spectrometry
LCꢀNSI+ꢀHRMS/MS ꢀ liquid chromatographyꢀpositive nanoelectrospray ionizationꢀhigh resolution tandem mass
spectrometry
[15N5]N6ꢀPHBꢀdAdo – [15N5]N6ꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyadenosine
N6ꢀPHBꢀdAdo – N6ꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyadenosine
N6ꢀPOBꢀdAdo – N6ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyadenosine
N1ꢀPHBꢀdIno – N1ꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyinosine
N1ꢀPOBꢀdIno – N1ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyinosine
NCS ꢀ N'ꢀchlorosuccinimide
NNK – 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone
NNAL ꢀ 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanol
NNKOAc – 4ꢀ(acetoxymethylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone
O6ꢀMeꢀdGuo – O6ꢀmethylꢀ2'ꢀdeoxyguanosine
O2ꢀPHBꢀdThd – O2ꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀthymidine
O2ꢀPOBꢀdThd – O2ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀthymidine
O2ꢀPOBꢀCyt – O2ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀcytosine
O6ꢀPHBꢀdG – O6ꢀ(4ꢀhydroxyꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyguanosine
O6ꢀPOBꢀdG – O6ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyguanosine
POB – pyridyloxobutyl
PHB – pyridylhydroxybutyl
[PyridineꢀD4]N6ꢀPOBꢀdAdo – [PyridineꢀD4]N6ꢀ(4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀ1ꢀbutyl)ꢀ2'ꢀdeoxyadenosine
SRM – selected reaction monitoring
TBAF – tetraꢀnꢀbutylammonium fluoride
TBSꢀCl – tertꢀbutyldimethylsilyl chloride
TFA – trifluoroacetic acid
THF ꢀ tetrahydrofuran
TMEDA ꢀ tetramethylethylenediamine
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pyridyl)ꢀ1ꢀbutanone and enantiomers of its metabolite 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanol in Fꢀ344 rats.
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Table 1: Levels of DNA adducts in lung and liver DNA from rats chronically treated with NNK, (S)ꢀNNAL, or (R)ꢀ
NNAL (5 ppm in their drinking water) for 50 weeks. Values are the average of three replicates and their standard
deviations.
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9
N6-POB-dAdo
(fmol/mg DNA)
O6-POB-dGuo
(fmol/mg DNA)
7-POB-Gua
(fmol/mg DNA)
O2-POB-Thd
(fmol/mg DNA) (fmol/mg DNA)
1531 ± 161a
< LOQ
O2-POB-Cyt
Tissue
Treatment
Liver
NNK
32 ± 9
27 ± 6
4 ± 1
< LOQa
< LOQ
< LOQ
9 ± 2
257 ± 32a
356 ± 18
28 ± 5
< LOQ
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(S)-NNAL
(R)-NNAL
NNK
1680 ± 100
< LOQ
< LOQ
< LOQ
< LOQ
130 ± 27
4409 ± 320
3951 ± 328
584 ± 45
O2-PHB-Thd
(fmol/mg DNA)
Lungb
99 ± 3
130 ± 1
16 ± 7
688 ± 65
568 ± 68
136 ± 10
(S)-NNAL
(R)-NNAL
10 ± 5
< LOQ
O6-PHB-dGuo
(fmol/mg DNA)
N6-PHB-dAdo
(fmol/mg DNA)
7-PHB-Gua
(fmol/mg DNA)
Liver
NNK
6 ± 1
5 ± 1
< LOQ
< LOQ
< LOQ
< LOQ
< LOQ
< LOQ
295 ± 42
133 ± 15
436 ± 23
129 ± 34
100 ± 25
379 ± 19
658 ± 107
422 ± 36
(S)-NNAL
(R)-NNAL
NNK
24 ± 2
35 ± 5
36 ± 8
125 ± 34
2995 ± 121
1259 ± 66
1152 ± 41
6291 ± 885
Lungb
(S)-NNAL
(R)-NNAL
Selected data are from Balbo et al (2014)18
DNA adduct data other than N6ꢀdAdo is from Ma et al (2017)19
a
b
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Figure Legends:
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5
Figure 1: Structures of previously measured POBꢀ and PHBꢀDNA adducts in vivo.
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Figure 2: Representative LCꢀESI+ꢀMS/MS chromatograms for in vitro formation of N6ꢀPOBꢀdA and N1ꢀPOBꢀdI. Samples
containing 25 mM K2PO3 buffer with esterase, esterase + DNA, or esterase + DNA + NNKOAc were incubated at 37 °C
for 24 h. Samples were analyzed after DNA hydrolysis and purification by solidꢀphase extraction (SPE). In each case, the
top two channels are monitoring separate transitions for the N1ꢀdIno adducts (POB: m/z 400 → m/z 284, 148; PHB: m/z
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402 → m/z 286, 132). The bottom two channels are monitoring the N6ꢀdAdo adducts (POB: m/z 399 → m/z 265; PHB: m/z
401 → m/z 132) and its isotopicallyꢀlabeled internal standard (POB: m/z 403 → m/z 269; PHB: m/z 406 → m/z 132).
Figure 3: Representative chromatograms obtained from the LCꢀESI+ꢀMS/MS analyses of (A) POBꢀ and (B) PHBꢀDNA
adducts in the lungs of rats chronically treated with 5 ppm NNK for 50 weeks. In each case, the top two channels are
monitoring separate transitions for the N1ꢀdIno adducts (POB: m/z 400 → m/z 284, 148; PHB: m/z 402 → m/z 286, 132).
The bottom two channels are monitoring the N6ꢀdAdo adducts (POB: m/z 399 → m/z 265; PHB: m/z 401 → m/z 132) and
its isotopicallyꢀlabeled internal standard (POB: m/z 403 → m/z 269; PHB: m/z 406 → m/z 132).
Figure 4: Levels of N6ꢀPOBꢀdA and N6ꢀPHBꢀdA in the liver and lung DNA of rats treated with NNKꢀ, (S)ꢀNNAL, and
(R)ꢀNNAL at a dose of 5 ppm in their drinking water for 50 weeks. Values are the average of three replicates and error
bars denote their standard deviation. POBꢀ and PHBꢀadducts are represented in blue and red, respectively. Liver and lung
tissue is differentiated by light and dark hues, respectively.
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