Pyridyloxobutyl adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase.

Article abstract of DOI:10.1021/tx9602067

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.

Full text of DOI:10.1021/tx9602067

562
Chem. Res. Toxicol. 1997, 10, 562-567
P yr id yloxobu tyl Ad d u ct
O6-[4-Oxo-4-(3-p yr id yl)bu tyl]gu a n in e Is P r esen t in
4-(Acetoxym eth yln itr osa m in o)-1-(3-p yr id yl)-1-bu ta n on e-Tr ea ted
DNA a n d Is a Su bstr a te for O6-Alk ylgu a n in e-DNA
Alk yltr a n sfer a se
Lijuan Wang,†,∇ Thomas E. Spratt,‡ Xiao-Keng Liu,†,| Stephen S. Hecht,†,
Anthony E. Pegg,§ and Lisa A. Peterson*,†,∇
Divisions of Chemical Carcinogenesis and of Pathology and Toxicology, American Health
Foundation, 1 Dana Road, Valhalla, New York 10595, and Department of Cellular and Molecular
Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033
Received December 17, 1996X
The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to
reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated
that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-
alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA
was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2′-
deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable
at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100
°C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 °C), O6-pobdG was
depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed
to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8
N HCl, 80 °C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the
model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone
([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or
recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the
pyridyloxobutyl group from the O6-position of guanine to AGT’s active site.
the tumorigenic activity of DNA methylation apparently
by increasing the levels and persistence of O6-mG in lung
DNA (6). Therefore, we have postulated that pyridyl-
oxobutyl adducts interfere with O6-mG repair, resulting
in increased persistence of this promutagenic adduct.
In tr od u ction
The tobacco-specific nitrosamine 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone (NNK1 ) selectively causes lung
tumors in laboratory animals (1-3). R-Hydroxylation of
this asymmetric nitrosamine forms two DNA reactive
species. Methyl hydroxylation generates pyridyloxobutyl
DNA adducts, whereas methylene hydroxylation leads
to methyl DNA adducts (4-6) (Scheme 1). Formation
and persistence of O6-methylguanine (O6-mG) is critical
for lung tumor induction in NNK-treated A/J mice (6).
The repair protein O6-alkylguanine-DNA alkyltrans-
ferase (AGT) repairs O6-mG in a reaction where the
methyl group is transferred from the O6-position of
guanine to an active site cysteinyl residue (7). Studies
in A/J mice indicated that pyridyloxobutylation enhanced
Pyridyloxobutylated DNA is capable of interfering with
O6-mG repair in vitro using rat liver AGT (8, 9). The
instability of pyridyloxobutyl adducts to chemical or
enzymatic hydrolysis of DNA complicated the precise
chemical characterization of the AGT substrate adducts.
The majority of these adducts decompose to 4-hydroxy-
1-(3-pyridyl)-1-butanone (HPB, 4; Scheme 1) upon enzy-
matic or strong acid hydrolysis of DNA (5, 10). Previous
studies indicated that a (pyridyloxobutyl)guanine adduct
was responsible for the AGT reactivity of this DNA (9).
Since mammalian AGT primarily reacts with O6-alkyl-
guanine residues in DNA, an O6-(pyridyloxobutyl)gua-
nine adduct is a possible AGT substrate adduct (Scheme
2). The model pyridyloxobutylating agent 4-(acetoxy-
methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc,
1) hydrolyzes to the corresponding R-hydroxynitrosamine
metabolite in the presence of esterase. This compound
decomposes to 4-oxo-4-(3-pyridyl)-1-butane diazohydrox-
ide (3) (11) (Scheme 1). The diazonium ion derived from
this species can react directly with guanine at the O6-
position to form an open-chain adduct or indirectly via a
cyclic oxonium ion (2) to form a cyclic adduct. Therefore,
there are two possible O6-(pyridyloxobutyl)guanine struc-
tures: an open-chain adduct, O6-[4-oxo-4-(3-pyridyl)butyl]-
* To whom correspondence should be addressed. Phone: (914) 789-
7229. Fax: (914) 592-6317.
† Division of Chemical Carcinogenesis.
‡ Division of Pathology and Toxicology.
§ Milton S. Hershey Medical Center.
∇ Current address: Division of Carcinogenesis and Molecular
Epidemiology, American Health Foundation.
| Current address: AMBI Inc., Tarrytown, NY 10591.
Current address: University of Minnesota Cancer Center, Min-
neapolis, MN 55455.
X Abstract published in Advance ACS Abstracts, April 15, 1997.
1 Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; DTT,
dithiothreitol; HEPES, N-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; NNK, 4-(methylnitro-
samino)-1-(3-pyridyl)-1-butanone; NNKOAc, 4-(acetoxymethylnitro-
samino)-1-(3-pyridyl)-1-butanone; O6-bzdG, 2′-deoxy-O6-benzylgua-
nosine; O6-pobdG, 2′-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine; O6-
pobG, O6-[4-oxo-4-(3-pyridyl)butyl]guanine; O6-mG, O6-methylguanine;
TCA, trichloroacetic acid.
S0893-228x(96)00206-8 CCC: $14.00 © 1997 American Chemical Society

Characterization of O6-Pyridyloxobutylguanine Adduct
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 563
Sch em e 1. P r op osed Activa tion P a th w a ys for NNK a n d NNKOAc
Sch em e 2. P r op osed Mech a n ism of Rea ction of Tw o P oten tia l O6-(P yr id yloxobu tyl)gu a n in e Ad d u cts w ith
AGT
guanine (5, O6-pobG), and a cyclic adduct, O6-[2-(3-
pyridyl)-2,3,4,5-tetrahydrofuran-2-yl]guanine (6; Scheme
2). Either one of these adducts will decompose under
hydrolysis conditions to generate HPB.
In this paper, the preparation and characterization of
2′-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG,
9) are described. The stability of this compound was
investigated. O6-[4-Oxo-4-(3-pyridyl)butyl]guanine (O6-
pobG) was detected in NNKOAc-treated DNA and was a
substrate for rat liver and human AGT.
Exp er im en ta l P r oced u r es
Ca u tion : NNKOAc is mutagenic as well as carcinogenic in
laboratory animals.

564 Chem. Res. Toxicol., Vol. 10, No. 5, 1997
Wang et al.
Ma ter ia ls. NNKOAc, [5-3H]NNKOAc (specific activity, 850
mCi/mmol), 2′-deoxy-O6-benzylguanosine, 2′-deoxy-N2-isobu-
tyrylguanosine 3′,5′-diisobutyrate, and [3H]methylated DNA
were prepared as previously described (8, 11-14). 3-[2-(3-
Pyridyl)-1,3-dithian-2-yl]propan-1-al was reduced with NaBH4
in MeOH to yield 3-[2-(3-pyridyl)-1,3-dithian-2-yl]propan-1-ol
(15). [5-3H]NNKOAc-treated DNA was prepared as previously
reported (8) and dialyzed against water overnight prior to use.
Rat liver AGT was obtained according to published procedures
(9). Human AGT with a polyhistidine tag at the amino terminus
was expressed and purified as previously reported (16). Calf
thymus DNA and porcine liver esterase were purchased from
Sigma Chemical Co. (St. Louis, MO).
M-1 cm-1), 281 (ꢀ ) 7650). See Supporting Information for 1H
NMR spectrum.
2′-Deoxy-O6-[4-oxo-4-(3-p yr id yl)b u t yl]gu a n osin e (O6-
p obd G, 9). A solution of 8 (10 mg, 0.02 mmol) in acetonitrile
(0.2 mL) was added in one portion to a well-stirred solution of
N-chlorosuccinimide (10.4 mg, 0.08 mmol) and silver nitrate (15
mg, 0.088 mmol) in 80% aqueous acetonitrile (1 mL) at 25 °C
(19). After 20 min, saturated aqueous solutions of Na2SO3,
Na2CO3, and NaCl were successively added (0.2 mL each). Then
CH2Cl2 (5 mL) was added, and the mixture was filtered through
Celite. After the filter cake was washed thoroughly with
CH2Cl2, the organic filtrate was dried with anhydrous MgSO4
and filtered. Compound 9 was purified by preparative TLC,
eluting with toluene/ethyl acetate/MeOH (10:8:3) (7.4 mg, 18
µmol, 90% yield, >95% pure): 1H NMR (CDCl3) δ 9.18 (d, J )
1.9 Hz, 1H, 2-pyridyl), 8.77 (dd, J ) 4.8, 1.7 Hz, 1H, 6-pyridyl),
8.24 (dt, J ) 8.0, 1.9 Hz, 1H, 4-pyridyl), 7.62 (s, 1H, C8-H), 7.41
(dd, J ) 7.9, 4.8 Hz, 1H, 5-pyridyl), 6.70 (bs, 1H, 5′-OH), 6.22
(dd, J ) 9.6, 5.5 Hz, 1H, 1′-H), 4.86 (s, 2H, N2-H2), 4.77 (d, J )
4.9 Hz, 1H, 3′-H), 4.62 (t, J ) 6.0 Hz, 2H, O6-CH2), 4.20 (s, 1H,
5′-Ha), 3.98 (dd, J ) 12.8, 1.5 Hz, 1H, 4′-H), 3.77 (d, J ) 12.7
Hz, 1H, 5′-Hb), 3.47 (s, 1H, 3′-OH), 3.24 (t, J ) 7.0 Hz, 2H, O6-
CH2CH2CH2CO), 3.08-3.00 (m, 1H, 2′-Ha), 2.36-2.29 (m, 2H,
O6-CH2CH2), 2.25 (dd, J ) 13.5, 5.5 Hz, 1H, 2′-Hb); UV λmax
(MeOH) 234 nm (ꢀ ) 14 100 M-1 cm-1), 275 (ꢀ ) 10 400); positive
ESI-MS/MS m/ z (rel intensity) 415 (M + 1, 60), 299 (M - 2′-
deoxyribose, 100), 268 (M - pyridyloxobutyl, 38), 152 [M -
(pyridyloxobutyl + 2′-deoxyribose), 50], 148 (M - 2′-deoxygua-
nosine, 55); high-resolution FAB MS calcd for C19H22N6O5
415.1727, found 415.1734. See Supporting Information for 1H
NMR spectrum.
O6-[4-Oxo-4-(3-p yr id yl)b u t yl]gu a n in e (O6-p ob G). O6-
pobdG (2 mg, 4.8 µmol) was added to 0.1 N HCl (5 mL) and
heated at 80 °C for 30 min. After cooling to room temperature,
the mixture was neutralized with 0.1 N NaOH (5 mL) and
applied to a C18 Sep-pak cartridge (Waters Corp., Milford, MA).
The product was eluted with H2O:MeOH (1:1) and identified as
O6-pobG: 1H NMR (DMSO-d6) δ 12.55 (s, 1H, N9H), 9.14 (d, J
) 1.5 Hz, 2-pyridyl), 8.78 (dd, J ) 4.8, 1.5 Hz, 1H, 4-pyridyl),
8.33-8.31 (m, 1H, 6-pyridyl), 7.78 (s, 1H, C8-H), 7.56 (dd, J )
7.9, 4.9 Hz, 1H, 5-pyridyl), 6.20 (s, 2H, N2H2), 4.45 (t, J ) 6.6
Hz, 2H, O6-CH2), 3.26 (t, J ) 6.8 Hz, 2H, O6-CH2CH2CH2), 2.15-
2.09 (m, 2H, O6-CH2CH2); positive ESI-MS/MS m/ z (rel inten-
sity) 299 (M + 1, 90), 152 (M - pyridyloxobutyl, 50), 148 (M -
guanine, 100); UV λmax (1:1 MeOH:H2O) 276 nm.
Sta bility Stu d ies. (a) O6-pobdG (1.1 mM) was incubated
in 50 mM HEPES (pH 7.8) in the presence or absence of 1.2
mM DTT for 30 min at 37 °C. (b) O6-pobdG (1 mM) was heated
in 10 mM sodium cacodylate (pH 7.0) at 100 °C for 60 min prior
to HPLC analysis. (c) O6-pobdG (1 mM) in 10 mM sodium
cacodylate (pH 7.0) and 10 mM sodium azide were incubated
at 37 °C for up to 13 days prior to HPLC analysis. Following
the addition of 0.1 vol of 1 N HCl, O6-pobdG (1 mM) in 10 mM
sodium cacodylate (pH 7.0) was heated at 80 °C for 30 min prior
to HPLC analysis. (d) Finally, the stability of O6-pobdG was
also determined under strong acid hydrolysis conditions (0.8 N
HCl, 80 °C, 1 h). All mixtures were analyzed by HPLC using a
Phenomenex Bondaclone C18 column (300 × 3.9 mm; Torrance,
CA) eluted with solvent A (20 mM sodium phosphate, pH 7.0)
and solvent B (95% methanol) with a linear gradient from 100%
solvent A to 50% solvent A/50% solvent B over 20 min followed
by a 10 min hold at 50% solvent A/50% solvent B (flow rate, 1
mL/min). The retention times (min) of the compounds are as
follows: guanine (11.0), HPB (20.0), O6-pobG (26.6), and O6-
pobdG (27.2).
DNA Hyd r olysis. [5-3H]NNKOAc-treated DNA was heated
in 0.1 N HCl at 80 °C for 0.5 h and frozen at -20 °C until
analysis. The sample was neutralized with 1 M potassium
phosphate (pH 7.4, 0.2 mL) prior to analysis by HPLC with
radioflow detection. The mixture was separated on a Phenom-
enex Bondaclone C18 column (300 × 3.9 mm; Torrance, CA) with
a linear gradient from 100% solvent A to 50% solvent A/50%
solvent B over 60 min (flow rate, 1 mL/min). The retention time
In str u m en ta l An a lyses. NMR spectra were acquired with
a Bruker Model AM360 WB spectrometer and are reported in
ppm relative to an external standard. UV spectra were collected
on a Hewlett Packard 8425A diode array spectrophotometer,
which was computer controlled by HP 89530 MS-DOS UV-vis
operating software. HPLC analyses were carried out with a
Waters 510 system (Millipore, Waters Division, Milford, MA)
with a Shimadzu SPD-UV-vis detector or â-Ram radioflow
detector (IN/US Systems, Inc., Tampa, FL). Electrospray MS
analyses were obtained on a Finnigan Model TSQ 700 tandem
quadruple mass spectrometer with a LC interface.
2′-Deoxy-N2-isobu tyr yl-O6-[3-[2-(3-p yr id yl)-1,3-d ith ia n -
2-yl]p r op yl]gu a n osin e 3′,5′-Diisobu tyr a te (7). 2′-Deoxy-N2-
isobutyrylguanosine 3′,5′-diisobutyrate (0.239 g, 0.5 mmol),
triphenylphosphine (0.197 g, 0.75 mmol), and 4-(1,3-dithian-2-
yl)-4-(3-pyridyl)butan-1-ol (0.191 g, 0.75 mmol) were combined,
dried over P2O5 under vacuum for 1-2 days, and dissolved in
10 mL of anhydrous 1,4-dioxane. Diethyl azodicarboxylate
(0.131 g, 0.75 mmol) in anhydrous 1,4-dioxane (2 mL) was added
dropwise at room temperature (17, 18). After 18 h, the reaction
mixture was concentrated under reduced pressure. CH2Cl2 (250
mL) was added, and the solution was washed twice with H2O,
dried over MgSO4, and filtered. The product was purified by
preparative TLC (PZF 254S, 20 × 20 cm, 1 mm thick; EM
Separations Technology, Gibbstown, NJ ) with elution by
CH2Cl2/ethyl acetate (1:1). The lowest band contained 7 (268
mg, 0.38 mmol, 75% yield, >95% pure): 1H NMR (CDCl3) δ 9.22
(d, J ) 1.9 Hz, 1H, 2-pyridyl), 8.55 (dd, J ) 4.9, 1.1 Hz, 1H,
6-pyridyl), 8.46 (d, J ) 8.1 Hz, 1H, 4-pyridyl), 8.23 (s, 1H, N2-
H), 7.95 (s, 1H, C8-H), 7.49 (dd, J ) 8.1, 5.0 Hz, 1H, 5-pyridyl),
6.37 (dd, J ) 8.0, 6.1 Hz, 1H, 1′-H), 5.42-5.41 (m, 1H, 3′-H),
4.49-4.30 (m, 5H, O6-CH2, 5′-H, 4′-H), 3.00-2.55 [m, 9H, 2′-
CH2, SCH2CH2CH2S, (CH3)2CH], 2.25-2.20 (m, 2H, O6-CH2-
CH2CH2), 1.99-1.86 (m, 4H, SCH2CH2CH2S, O6-CH2CH2),
1.29-1.13 [m, 18H, (CH3)2CH]; positive ESI-MS/MS m/ z (rel
intensity) 715 (M + 1, 100), 238 (M - 2′-deoxy-N2-isobu-
tyrylguanosine 3′,5′-diisobutyrate, 90). See Supporting Infor-
mation for 1H NMR spectrum.
2′-Deoxy-O6-[3-[2-(3-pyr idyl)-1,3-dith ian -2-yl]pr opyl]gu a-
n osin e (8). NaOH (2 N, 10 mL) was added to 7 (30 mg, 0.042
mmol) in MeOH (10 mL) at room temperature. After 3 h, the
pH was adjusted to 7 with 80% aqueous acetic acid, and the
reaction mixture was extracted with ethyl acetate. The organic
layers were combined, washed with H2O, dried over MgSO4, and
filtered. Compound 8 was purified by preparative TLC, eluting
with toluene/ethyl acetate/MeOH (10:8:3) (18 mg, 0.036 mmol,
85% yield, >95% pure): 1H NMR (CDCl3) δ 9.14 (d, J ) 2.2 Hz,
1H, 2-pyridyl), 8.49 (dd, J ) 4.7, 1.4 Hz, 1H, 6-pyridyl), 8.21
(dt, J ) 8.1, 2.1 Hz, 1H, 4-pyridyl), 7.61 (s, 1H, C8-H), 7.29 (dd,
J ) 8.1, 4.7 Hz, 1H, 5-pyridyl), 6.69 (d, J ) 11.1 Hz, 1H, 5′-
OH), 6.22 (dd, J ) 9.4, 5.6 Hz, 1H, 1′-H), 4.96 (s, 2H, N2-H2),
4.75 (bd, J ) 4.6 Hz, 1H, 3′-H), 4.36 (t, J ) 6.6 Hz, 2H, O6-
CH2), 4.20 (s, 1H, 5′-Ha), 3.96 (d, J ) 12.1 Hz, 1H, 4′-H), 3.75 (t,
J ) 11.4 Hz, 1H, 5′-Hb), 3.03-2.96 (m, 2H, 3′-OH, 2′-Ha), 2.73-
2.59 (m, 4H, SCH2CH2CH2S), 2.26 (dd, J ) 13.3, 5.6 Hz, 1H,
2′-Hb), 2.21-2.16 (m, 2H, O6-CH2CH2CH2), 2.00-1.79 (m, 4H,
O6-CH2CH2, SCH2CH2CH2S); positive ESI-MS/MS m/ z (rel
intensity) 505 (M + 1, 100), 389 (M - 2′-deoxyribose, 51), 238
(M - 2′-deoxyguanosine, 49); λmax (MeOH) 250 nm (ꢀ ) 8700

Characterization of O6-Pyridyloxobutylguanine Adduct
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 565
Sch em e 3. Syn th etic Rou te to O6-p obd Ga
a
Ib ) isobutyryl, Ph3P ) triphenylphosphine, DEAD ) diethyl azodicarboxylate, NCS ) N-chlorosuccinimide.
of O6-pobG in this system was 57 min. Total levels of HPB-
releasing adducts were determined as previously described (6).
O6-pobG and HPB levels were expressed relative to the guanine
concentrations of the same samples (4).
The radioactive peak that coeluted with O6-pobG standard
was collected using a linear gradient from 100% A to 50% A/50%
B over 50 min. A portion of this fraction was reacted with
NaBH4 and reanalyzed by HPLC with radioflow detection. HCl
was added to another portion of this fraction (final concentra-
tion, 0.8 N HCl). This solution was heated at 80 °C for 4 h and
reanalyzed following neutralization by HPLC with radioflow
detection. The retention times (min) of the compounds were
HPB (34.0), O6-pobG (50.0), and O6-pobdG (53.5).
In cu ba tion of O6-p obd G w ith AGT. O6-pobdG (0.9 mM)
or O6-benzyl-2′-deoxyguanosine (O6-bzdG; 0.7 mM) was incu-
bated with rat liver AGT (0.67 pmol) in the presence or absence
of calf thymus DNA (20 µg) in 50 mM HEPES (pH 7.8), 1 mM
DTT, and 1 mM EDTA (total volume, 1 mL). The nucleosides
were previously dissolved in the buffer at their maximum
solubility (O6-pobdG, 1.1 mM; and O6-bzdG, 0.9 mM). After 30
min at 37 °C, [3H]methylated DNA was added, and the incuba-
tions were continued for an additional 30 min at 37 °C. AGT
activity was determined as previously described (9).
In cu ba tion of [5-3H]NNKOAc-Tr ea ted DNA w ith AGT.
[5-3H]NNKOAc-treated DNA was incubated with either semi-
purified rat liver AGT (9 pmol) or purified human AGT with a
polyhistidine tag (6 pmol) in 35 mM HEPES, 1 mM DTT, 1 mM
EDTA, and 5% glycerol (pH 7.8) for 30 min at 37 °C (total
volume, 1 mL). Controls were performed either in the presence
of boiled rat liver protein or in the absence of AGT. The reaction
was stopped upon addition of bovine serum albumin (0.8 mg)
and 1.0 N HCl (0.1 mL). The resulting mixture was heated at
80 °C for 30 min. Following centrifugation, the supernatant
was removed, filtered, and analyzed by HPLC linked with
radioflow detection (see above). O6-pobG and HPB levels are
expressed relative to guanine concentrations of the same
samples.
the assigned structure. The stability of this compound
was examined under a variety of conditions. Incubation
of O6-pobdG in the AGT assay buffer demonstrated that
the compound was stable to the AGT reaction conditions
(pH 7.8, 37 °C for 30 min). Subsequent studies demon-
strated that this derivative was stable in pH 7 buffer at
37 °C for at least 13 days. It was also stable at 100 °C
for 30 min. When O6-pobdG was heated in 0.1 N HCl
for 30 min, a new peak appeared in the HPLC trace that
eluted immediately before O6-pobdG. Preparative isola-
tion and characterization by NMR, MS, and UV analyses
demonstrated that this new compound was O6-pobG.
When O6-pobdG was heated at 80 °C in 0.8 N HCl, it
decomposed to HPB and guanine with the intermediate
formation of O6-pobG. O6-pobdG was not stable under
basic conditions. A new compound was formed that did
not coelute with any of the standards.
With the knowledge that O6-pobdG depurinates to O6-
pobG under 0.1 N HCl hydrolysis conditions, we per-
formed similar hydrolyses of [5-3H]NNKOAc-treated
DNA to determine if this adduct was present. HPLC
analysis of the hydrolysates with radioflow detection
indicated the presence of a radioactive peak that coeluted
with the O6-pobG standard (Figure 1A). Following reac-
tion with NaBH4, the standard and the radioactive peak
eluted 4 min earlier, consistent with reduction. In
addition, the adduct released radioactivity that coeluted
with HPB when it was heated in 0.8 N HCl at 80 °C for
4 h. These results are consistent with the presence of
O6-pobG in the NNKOAc-treated DNA. Adduct levels in
the 0.1 N HCl hydrolysates were 5 ( 1 pmol of O6-pobG/
µmol of guanine and 42 ( 8 pmol of HPB-releasing
adducts/µmol of guanine (n ) 6). This compares to 73
pmol of HPB-releasing adducts/µmol of guanine mea-
sured in 0.8 N HCl hydrolysates (n ) 2).
Resu lts
O6-pobG was removed from [5-3H]NNKOAc-treated
DNA by AGT. Weak acid hydrolysates of [5-3H]-
NNKOAc-treated DNA that had been incubated with
partially purified rat liver AGT lacked the radioactive
The postulated O6-(pyridyloxobutyl)guanine DNA ad-
duct O6-pobdG (9) was prepared as outlined in Scheme
3. 1H NMR, UV, and MS analyses were consistent with

566 Chem. Res. Toxicol., Vol. 10, No. 5, 1997
Wang et al.
different stabilities. It is also possible that the cyclic
adduct 6 represents a greater percentage of the AGT
substrate adducts in NNKOAc-treated DNA. This ad-
duct is expected to have lower stability under hydrolysis
conditions (20).
O6-pobG in DNA was repaired by rat and human AGT,
demonstrating that it is a substrate for this repair
protein. Therefore, it can potentially compete with O6-
mG for reaction with this protein. These studies indicate
that the ability of NNKOAc-treated DNA to interfere
with repair of O6-mG is through a competition between
these two adduct types for reaction with AGT. Our
previous studies indicate that approximately 3-30% of
the original HPB-releasing adduct level are AGT sub-
strate adducts, depending on the DNA preparation (8,
9). O6-pobG represented 7% of the total HPB-releasing
adducts in the present study. At this time, we cannot
exclude the possibility that there are other potential AGT
substrate adducts in NNKOAc-treated DNA.
AGT only reacts with O6-pobG when it is incorporated
into DNA; O6-pobdG was unable to interfere with AGT’s
ability to repair O6-mG. This observation is consistent
with previous reports on the reactivity of other O6-
alkylguanine derivatives. AGT reacts more quickly with
O6-alkylguanine adducts in double-stranded DNA than
as free bases or nucleosides (21-27). The binding of AGT
to DNA is an important step in the initiation of adduct
repair and likely places the adduct in a better position
for reaction with the active site cysteine (23, 27).
The removal of O6-pobG from DNA by AGT indicates
that the pyridyloxobutyl group is transferred from the
O6-position of guanine to the cysteine residue at AGT’s
active site. This transfer can be effected either via direct
SN2 displacement or via the involvement of an oxonium
ion intermediate generating an alkylated protein (Scheme
2). The cyclic adduct 6 could also undergo similar
reactions. We have proposed that the transfer reaction
may be facilitated by neighboring group participation of
the carbonyl oxygen. Future studies will examine the
mechanism of pyridyloxobutyl group transfer.
F igu r e 1. Representative HPLC radiograms of 0.1 N HCl
hydrolysates of [5-3H]NNKOAc-treated DNA previously incu-
bated with (A) boiled rat liver AGT or (B) active rat liver AGT.
peak that coeluted with O6-pobG (Figure 1B). O6-pobG
levels were reduced to the limits of detection (less than
1.5 pmol of O6-pobG/µmol of guanine), demonstrating that
the adduct is a substrate for the repair protein. Controls
were performed in the presence of heat-treated protein
or bovine serum albumin. Similar results were obtained
with purified human AGT with a polyhistidine tag,
confirming that O6-pobG is a substrate for AGT.
High background complicated attempts to measure
radioactivity associated with the protein. The nucleoside
O6-pobdG was not active toward rat liver AGT under
conditions that yielded complete inhibition of the protein
by O6-bzdG.
Ack n ow led gm en t. We would like to thank the AHF
Research Animal Facility for animal treatment and tissue
isolation. High-resolution MS analyses were performed
by the Washington University Resource for Biomedical
and Bio-organic Mass Spectrometry, St. Louis, MO. This
research was supported by grants CA-59887 (L.A.P.), CA-
44377 (S.S.H.), and CA-18137 (A.E.P.) from the National
Cancer Institute. The Research Animal Facility and the
Instrument Facility at AHF are partially supported by
National Cancer Institute Cancer Center Support Grant
CA-17613.
Discu ssion
In this paper we report the first identification of a
pyridyloxobutyl DNA adduct, O6-pobG (9), in NNKOAc-
treated calf thymus DNA. The presence of other radioac-
tive peaks in the 0.1 N HCl hydrolysates suggests that
there are other adducts that are also stable to these
hydrolysis conditions. They are possibly depurinated
guanine or adenine adducts.
The stability of O6-pobdG under neutral thermal hy-
drolysis conditions was unexpected. AGT substrate
adducts in NNKOAc-treated calf thymus DNA were
removed from DNA during neutral thermal hydrolysis
(8). In contrast, the ability of NNKOAc-treated 19-mers
to react with AGT was stable to neutral thermal hydroly-
sis conditions (9). The reason for these differences is
unclear. However, it is possible that the adduct in
NNKOAc-treated DNA (generated as double-stranded
DNA) has an alternative conformation than that in the
oligomers (which were generated as single-stranded
DNA) and the nucleoside. These conformations may have
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 7-9 (3 pages). Ordering information is given on
any current masthead page.
Refer en ces
(1) Hecht, S. S., Chen, C. B., Ohmori, T., and Hoffmann, D. (1980)
Comparative carcinogenicity in F344 rats of the tobacco specific
nitrosamines, N′-nitrosonornicotine and 4-(N-methyl-N-nitro-
samino)-1-(3-pyridyl)-1-butanone. Cancer Res. 40, 298-302.
(2) Hecht, S. S., and Hoffmann, D. (1988) Tobacco-specific nitro-
samines: an important group of carcinogens in tobacco and
tobacco smoke. Carcinogenesis 9, 875-884.
(3) Hecht, S. S., Morse, M. A., Amin, S. G., Stoner, G. D., J ordan, K.
G., Choi, C.-I., and Chung, F.-L. (1989) Rapid single dose model

Characterization of O6-Pyridyloxobutylguanine Adduct
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 567
for lung tumor induction in A/J mice by 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone and the effect of diet. Carcinogenesis 10,
1901-1904.
an internal standard for analysis of tobacco-specific nitrosamine
hemoglobin and DNA adducts. J . Labelled Compd. Radiopharm.
33, 285-292.
(4) Hecht, S. S., Trushin, N., Castonguay, A., and Rivenson, A. (1986)
Comparative tumorigenicity and DNA methylation in F344 rats
by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-ni-
trosodimethylamine. Cancer Res. 46, 498-502.
(16) Edara, S., Kanugula, S., Goodtzova, K., and Pegg, A. E. (1996)
Resistance of the human O6-alkylguanine-DNA alkyltransferase
containing arginine at codon 160 to inactivation by O6-benzylgua-
nine. Cancer Res. 56, 5571-5575.
(5) Hecht, S. S., Spratt, T. E., and Trushin, N. (1988) Evidence for
4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats treated with
the tobacco specific nitrosamines 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone and N′-nitrosonornicotine. Carcinogenesis 9,
161-165.
(17) Himmelsbach, F., Schulz, B. S., and Trichtinger, T. (1984) The
p-nitrophenylethyl (NPE) group-a versatile new blocking group
for phosphate and aglycone protection in nucleosides and nucle-
otides. Tetrahedron Lett. 40, 59-72.
(18) Mitsunobu, O. (1981) The use of diethyl azodicarboxylate and
triphenylphosphine in synthesis and transformation of natural
products. Synthesis 1-28.
(19) Seebach, D., and Corey, E. J . (1975) Generation and synthetic
applications of 2-lithio-1,3-dithianes. J . Org. Chem. 40, 231-237.
(20) Carmella, S. G., Kagan, S. S., Spratt, T. E., and Hecht, S. S. (1990)
Evaluation of cysteine adduct formation in rat hemoglobin by
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and related com-
pounds. Cancer Res. 50, 5453-5459.
(21) Bhattacharyya, D., Foote, R. S., Boulden, A. M., and Mitra, S.
(1990) Physicochemical studies of human O6-methylguanine-DNA
methyltransferase. Eur. J . Biochem. 193, 337-343.
(22) Liem, L. K., Wong, C. W., Lim, A., and Li, B. F. (1993) Factors
influencing the repair of the mutagenic lesion O6-methylguanine
in DNA by human O6-methylguanine-DNA methyltransferase. J .
Mol. Biol. 231, 950-959.
(6) Peterson, L. A., and Hecht, S. S. (1991) O6-Methylguanine is a
critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone tumorigenesis in A/J mouse lung. Cancer Res. 51, 5557-
5564.
(7) Pegg, A. E. (1990) Properties of mammalian O6-alkylguanine-DNA
transferases. Mutat. Res. 233, 165-175.
(8) Peterson, L. A., Liu, X.-K., and Hecht, S. S. (1993) Pyridyloxobutyl
DNA adducts inhibit the repair of O6-methylguanine. Cancer Res.
53, 2780-2785.
(9) Liu, X. K., Spratt, T. E., Murphy, S. E., and Peterson, L. A. (1996)
Pyridyoxobutylation of guanine residues by 4-(acetoxymethylni-
trosamino)-1-(3-pyridyl)-1-butanone generates substrates of O6-
alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 9, 949-
953.
(10) Spratt, T. E., Trushin, N., Lin, D., and Hecht, S. S. (1989) Analysis
for N2-(pyridyloxobutyl)deoxyguanosine adducts in DNA of tissues
exposed to tritium labelled 4-(methylnitrosamino)-1-(3-pyridyl)-
1-butanone and N′-nitrosonornicotine. Chem. Res. Toxicol. 2, 169-
173.
(23) Goodtzova, K., Crone, T. M., and Pegg, A. E. (1994) Activation of
human O6-alkylguanine-DNA alkyltransferase by DNA. Biochem-
istry 33, 8385-8390.
(24) Dolan, M. E., Scicchitano, D., and Pegg, A. E. (1988) Use of
oligodeoxynucleotides containing O6-alkylguanine for the assay
of O6-alkylguanine-DNA-alkyltransferase activity. Cancer Res. 48,
1184-1188.
(25) Chan, C.-L., Wu, Z., Ciardelli, T., Eastman, A., and Bresnick, E.
(1993) Kinetic and DNA-binding properties of recombinant human
O6-methylguanine-DNA methyltransferase. Arch. Biochem. Bio-
phys. 300, 193-200.
(26) Pegg, A. E., Boosalis, M., Samson, L., Moschel, R. C., Byers, T.
L., Swenn, K., and Dolan, M. E. (1993) Mechanisms of inactivation
of human O6-alkylguanine-DNA alkyltransferase by O6-ben-
zylguanine. Biochemistry 32, 11998-12006.
(27) Goodtzova, K., Kanugula, S., Edara, S., Pauly, G. T., Moschel,
R., and Pegg, A. E. (1997) Repair of O6-benzylguanine by the E.
coli Ada and Ogt and the human O6-alkylguanine-DNA alkyl-
transferases. J . Biol. Chem., in press.
(11) Spratt, T. E., Peterson, L. A., Confer, W. L., and Hecht, S. S. (1990)
Solvolysis of model compounds for R-hydroxylation of N′-nitroso-
nornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-
one: Evidence for
a cyclic oxonium ion intermediate in the
alkylation of nucleophiles. Chem. Res. Toxicol. 3, 350-356.
(12) Peterson, L. A., Carmella, S. G., and Hecht, S. S. (1990) Investiga-
tions of metabolic precursors to hemoglobin and DNA adducts of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis
11, 1329-1333.
(13) Pauly, G. T., Powers, M., Pei, G. K., and Moschel, R. C. (1988)
Synthesis and properties of H-ras DNA sequences containing O6-
substituted 2′-deoxyguanosine residues at the first, second, or both
positions of codon 12. Chem. Res. Toxicol. 1, 391-397.
(14) Narang, S. A., Brousseau, R., Hsiung, H. M., and Michniewicz,
J . J . (1980) Chemical synthesis of deoxyoligonucleotides by the
modified triester method. Methods Enzymol. 65, 610-620.
(15) Lin, J .-M., Amin, S., Murphy, S. E., Solomon, J . J ., and Hecht, S.
S. (1993) Synthesis of [3,3-D2]4-hydroxy-1-(3-pyridyl)-1-butanone,
TX9602067