Products of the direct reaction of the diazonium ion of a metabolite of the carcinogen N -nitrosomorpholine with purines of nucleosides and dna

Article abstract of DOI:10.1021/tx100093a

A number of putative purine nucleoside and nucleobase adducts of the diazonium ion derived from 3-hydroxy-N-nitrosomorpholine have been synthesized as dimethylacetals. These are converted, in most cases nearly quantitatively, to the aldehydes, or in two c

Full text of DOI:10.1021/tx100093a

Chem. Res. Toxicol. 2010, 23, 1223–1233
1223
Products of the Direct Reaction of the Diazonium Ion of a
Metabolite of the Carcinogen N-Nitrosomorpholine with Purines of
Nucleosides and DNA
Charles N. Zink, Nicolas Soissons, and James C. Fishbein*
Department of Chemistry and Biochemistry, UniVersity of Maryland, Baltimore County, 1000 Hilltop Circle,
Baltimore, Maryland 21228
ReceiVed March 11, 2010
A number of putative purine nucleoside and nucleobase adducts of the diazonium ion derived
from 3-hydroxy-N-nitrosomorpholine have been synthesized as dimethylacetals. These are converted,
in most cases nearly quantitatively, to the aldehydes, or in two cases to their derivatives, on treatment
with mild acid to yield standards for a quantitative investigation of alkylation of purine nucleosides
and DNA by the above metabolite of the powerful carcinogen N-nitrosomorpholine. The stability of
the resulting nucleobase ethoxyacetaldehyde (EA) adducts has been characterized under a number
of conditions with respect to their propensity to decompose. The stabilities, compared to that of the
previously characterized adduct of the model benzimidazole, are generally unexceptional. Deposition
of adducts on purine nucleosides and DNA were quantified in reactions in which 3-hydroperoxy-
N-nitrosomorpholine was reduced to the hydroxy metabolite by a water-soluble phosphine at 21 (
2 °C. The adduct profile is highly similar to that observed from simpler R-hydroxy metabolites of
acyclic dialkylnitrosamines, with the three most abundant ethoxyacetaldehyde (EA) adducts in
reactions of duplex DNA being N7-EA-Gua ∼ O6-EA-Gua > N3-EA-Ade. The initial rate kinetics
of formation of hydroxyethyl (HE) lesions from the initially formed EA lesions have been determined
in the case of the major products in the cases of both the nucleoside and DNA adducts. The rates
of formation of HE adducts are accelerated in DNA, relative to the nucleosides in the cases of the
N7-EA-Ade, N7-EA-Gua, and O6-EA-Gua adducts by factors of 7, 14, and 54, respectively. The
initial rates of depurination of the N3-EA-Ade, N7-EA-Gua, and N7-EA-Gua adducts have also
been quantified, and they are unexceptional in comparison with what has been previously reported
for simple alkyl adducts. The adduct profiles reported here stand in significant contrast to what has been
reported previously for structurally closely related R-substituted cyclic nitrosamines. In part or whole, this
may be due to methodological differences in the conduct of the present and previous reports.
DNA damage profile for NMOR is marginally characterized.
Isolation of N7-hydroxyethyl-Gua (N7-HE-Gua) from rats
treated with NMOR was reported 30 years ago (16). A dGua
adduct derived from crotonaldehyde formed in the decay of the
diazoic in Scheme 1 in the presence of dGua has been isolated
(17).
1. Introduction
N-Nitrosomorpholine (NMOR), 1 Scheme 1, is a powerful
liver carcinogen in rats and mice and a respiratory tract
carcinogen in hamsters (1-3). NMOR is found in ground-
water, wastewater effluents, foods, cosmetics and toiletries
(4-6), petroleum based fluids, and some industrial workplace
airspaces, particularly in the rubber industry (7-10). NMOR
is one of seven nitrosamine carcinogens for which NIOSH
has specifically developed a valid analytical detection
methodology in the form of Protocol 2522 for workspace
monitoring (11). NMOR appears to be formed endogenously.
NMOR has been detected in human urine (12, 13) and human
gastric juices (14), and levels are elevated in individuals who
simultaneously consume nitrate-rich and protein-rich foods
(13).
There is good evidence from studies of the metabolism of
the radiolabeled compound that a significant part of the
metabolism involves simple R-hydroxylation, path a in Scheme
1, as indicated by the isolation of the metabolite, bottom of
Scheme 1 (18, 19). Largely through the efforts of the Loeppky
group, a second pathway involving ꢀ hydroxylation has been
elaborated (path b, Scheme 1) (20-26). This pathway gives
rise to an overlapping set of DNA damaging agents some of
which have been characterized. While it was reported earlier
that R-hydroxyNMOR, 2, yields “almost entirely” acetaldehyde
(27, 28), this was later demonstrated to be incorrect (29). About
one-quarter of the diazonium ion derived from R-hydroxyN-
MOR does undergo rearrangement (kr, Scheme 1) generating
acetaldehyde and glycoaldehyde. Predominantly though, R-hy-
droxyNMOR forms 2-hydroxyethoxyacetaldehyde or its cyclic
form by either the kH2O and kIM pathways, respectively, in
Scheme 1.
Though NMOR is widely used as model for the induction of
liver cancer in animal models, little is known of the mutation
or DNA damage spectrum from this compound. It has been
demonstrated that mutations in Hras are largely transversions
at both GC and AT sites, quite different from the textbook GC
f AT transitions that typify the simple nitrosamines (15). The
Anticipation that direct reaction of the diazonium ion in
Scheme 1 might give rise to DNA adducts, classical nitrosamine
* To whom correspondence should be addressed. Tel: 410-455-2190.
E-mail: jfishbei@umbc.edu.
10.1021/tx100093a  2010 American Chemical Society
Published on Web 05/05/2010

1224 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Scheme 1. Metabolic Fate of N-Nitrosomorphloline
Zink et al.
chemistry of the diazonium ion, which does not appear to
produce hydroxyethylating equivalents, with the observation 30
years ago, above, of the product 7-hydroxyethylGua (7-HE-
Gua) isolated from rats administered NMOR (16).
In the present article, a number of the putative purine-EA
nucleobase adducts have been synthesized as their stable
dimethyl acetals. Treatment of dilute solutions of these with
acid nearly quantitatively generates, in most cases, the alde-
hydes, chiefly in the hydrate form. These solutions are then
employed as standards for the analysis and quantitation of the
products of reactions of R-hydroxyNMOR, derived from hy-
droperoxide 3 (Scheme 1) with purine nucleosides and DNA,
subsequent to acid catalyzed depurination. The stability of the
EA moiety and/or its propensity toward conversion to HE
adducts on the purine nucleobases, alkylated nucleosides, and
in calf thymus DNA has been studied. Surprisingly, on the basis
of the dearth of diazonium ion adducts from other cyclic
nitrosamines, the spectrum of adducts and relative quantities
reflect what is observed for simple diazonium ions derived from
the R-hydroxy metabolites of acyclic dialkylnitrosamines. In
addition, it is observed that nucleobase-EA adducts are not of
exceptional reactivity, but in the cases of some adducts, duplex
DNA significantly accelerates the conversion to HE adducts.
2. Experimental Section
2.1. Chemicals. Except as noted, chemicals, solvents, and
isotopically enriched reagents employed in synthetic and analytical
procedures were obtained from readily available commercial
sources. Deionized water was obtained in house and filtered for
HPLC and LC/MS experiments. Solvents such as methylene
chloride, acetonitrile, ethyl acetate, and hexanes were dried and
distilled from calcium hydride. Chloroform was distilled from
sodium sulfate. Methanol and ethanol were distilled from magne-
sium turnings. Ether and tetrahydrofuran were dried and distilled
from sodium. Methanol and acetonitrile used in HPLC and LC/
MS experiments were purchased as HPLC grade solvents and were
not further purified.
damage, is tempered by the extensive studies of Hecht on the
DNA adducts from N-nitrosopyrrolidine (NPYR) and N-
nitrosopiperidine (NPIP) (30-40).
2.2. Analytical Determinations. Measurements of pH were
performed on an Orion pH meter, model SA720, with a combination
electrode. Two point calibrations were done before pH values were
recorded. Calibrations were performed using commercially available
standards. Elemental analyses were performed by Atlantic Micro-
labs, Inc., Norcross, GA. High resolution mass spectra were
obtained at the UMBC Center for Biological Mass Spectrometry.
Low resolution mass spectra and nucleobase adduct quantification
were performed on a Waters 2695 liquid chromatograph attached
to a Waters Micromass ZQ Mass Spectrometer.
Of the nine nucleoside adducts so far derived from reactions of
R-hydroxyNPYR with nucleosides or DNA, only two of these,
one being the most abundant among all the known adducts, are
claimed to be derived from direct reaction of the analogous
diazonium ion. To date, not a single detected nucleoside adduct
derived from reactions of R-hydroxyNPIP with nucleosides or
DNA involves direct reaction of the diazonium ion. In the case
of both NPYR and NPIP, the adducts not derived from direct
reaction with diazonium ions are due to electrophilic products
of diazonium ion decay.
2.3. Synthesis. The compounds (15N2)-2′-deoxyguanosine (42),
(15N6)-2′-deoxyadenosine (42-44), R-hydroperoxy-N-nitrosomor-
pholine (29, 41), 2-(6-amino-7H-purin-7-yl)ethanol (45), 2-(9H-
purin-6-ylamino)ethanol (46), 2-(6-amino-3H-purin-3-yl)ethanol
(47), 2-(2-amino-9H-purin-6-yloxy)ethanol (48), 2-amino-7-(2-
hydroxyethyl)-1H-purin-6(7H)-one (49), 9-((2R,4R,5R)-4-hydroxy-
5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-(2-hydroxyethylamino)-
1H-purin-6(9H)-one (50), and 2-amino-9-((2S,4S,5S)-4-hydroxy-
5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1-(2-hydroxyethyl)-1H-
purin-6(9H)-one (51, 52) were synthesized as described in the
references cited and exhibited 1H NMR and 13C NMR spectra
consistent with either what was previously reported or, in the
absence of such, the structure indicated. Hydroxyethylated nucleo-
bases from the last two compounds were liberated by acid hydrolysis
and purified by neutralization and recrystallization in water.
2.4. 2-(2,2-Dimethoxyethoxy)ethyl methanesulfonate (4). A so-
lution of 2-(2,2-dimethoxyethoxy)ethanol (53) (20.1 g, 0.134 mol)
in dry pyridine (40 mL) and dry CH2Cl2 (80 mL) was cooled to 0
°C under a nitrogen atmosphere. Methanesulfonyl chloride (15.5
mL, 0.201 mol) was added dropwise to the stirring solution. The
Notwithstanding, it was recently established that the diazo-
nium ion derived from R-hydroxyNMOR deposits an ethoxy-
acetaldehyde (EA) on the “model nucleobase” benzimidazole
presaging actual EA-nucleobase adducts, eq 1 (41).
It was additionally demonstrated that the EA-benzimidazole
adduct slowly decays to a hydroxyethyl (HE) adduct and that
this decay is accelerated by primary and secondary amines or
base (eq 1). This latter observation rationalizes the known

Direct Reaction of the Diazonium Ion with Purines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1225
solution was stirred for 1 h at 0 °C and then 2 h at room temperature.
The resulting suspension was then cooled to 0 °C and 150 mL of
H2O was added slowly. The solution was then extracted with
CH2Cl2 (3 × 200 mL). The organic extracts were combined, washed
sequentially with 1 M HCl (1× 100 mL), saturated NaHCO3 (1×
100 mL), and brine (1× 100 mL), and were then dried over MgSO4.
After filtration, the solvent was removed under reduced pressure
to yield a pale yellow liquid (90% yield) that was used without
further purification. 1H NMR (CDCl3) δ 3.04 (3H, s), 3.37 (6H, s),
3.52 (2H, d, J ) 5.0 Hz), 3.74-3.76 (2H, m), 4.34-4.36 (2H, m),
4.47 (1H, t, J ) 5.0 Hz). 13C NMR (CDCl3) δ 102.8, 71.1, 69.5,
69.2, 54.2, 37.9.
under a N2 atmosphere. To the suspension was added diisopropyl
azodicarboxylate (0.75 mL, 2.6 mmol). The solution was stirred at
0 °C for 10 min and then at room temperature for 18 h. The solution
was concentrated and purified by silica gel column chromatography
(100% diethyl ether) (Rf 0.27) to yield a clear liquid that crystallized
upon standing (65% yield). 1H NMR (CDCl3) δ 0.06 (6H, s), 0.08
(6H, s), 0.89 (18H, s), 2.33 (1H, m), 2.55 (1H, m), 3.37 (6H, s),
3.58 (2H, d, J ) 5.0 Hz), 3.70-3.80 (2H, m), 3.90 (2H, t, J ) 5.5
Hz), 3.95 (1H, m), 4.49 (2H, m), 4.56 (1H, m), 4.63 (2H, t, J )
5.0 Hz), 4.80 (2H, s), 6.30 (1H, t, J ) 6.9 Hz), 7.88 (1H, s).
2.9. (2R,3R,5R)-5-(2-Amino-6-(2-(2,2-dimethoxyethoxy)ethoxy)-
9H-purin-9-yl)-2-(hydroxymethyl)-tetrahydrofuran-3-ol (9). Com-
pound 8 (250 mg, 0.4 mmol), potassium fluoride (196 mg, 3.4
mmol), and 18-crown-6 (105 mg, 0.4 mmol) were dissolved in DMF
(5 mL) and stirred at 60 °C for 2 h. The solution was cooled to
room temperature and concentrated under reduced pressure. The
residue was dissolved in water (10 mL) and washed with CH2Cl2
(3 × 15 mL). The aqueous extract was collected, concentrated, and
purified by silica gel column chromatography (90:5:5 CH3CN/H2O/
NH4OH) (Rf 0.35) to yield a clear solid (90% yield).1H NMR
(DMSO) δ 2.21 (1H, m), 2.53 (1H, m), 3.26 (6H, s), 3.47 (2H, d,
J ) 5.0 Hz), 3.48-3.60 (2H, m), 3.78-3.82 (3H, m), 4.35 (1H,
m), 4.46-4.52 (3H, m), 4.99 (1H, t, J ) 5.5 Hz), 5.27 (1H, d, J )
4.1 Hz), 6.20 (1H, t, J ) 6.0 Hz), 6.44 (2H, s), 8.09 (1H, s).13C
NMR (DMSO) δ 160.2, 159.7, 154.0, 137.8, 113.9, 102.3, 87.6,
82.8, 70.8, 70.2, 68.9, 64.9, 61.7, 53.4, 39.4. Mass Calcd for
C16H26N5O7: 400.1833. Mass Obsd: 400.1827.
2.5. 7-(2-(2,2-Dimethoxyethoxy)ethyl)-7H-purin-6-amine (5). To
a solution of N-(4,6-diaminopyrimidin-5-yl)formamide (47) (500
mg, 3.3 mmol) in dry DMF (10 mL) was added sodium hydride
(130 mg, 3.3 mmol, 60% in mineral oil). The solution was heated
at 50 °C. After 30 min, a solution of 4 (782 mg, 3.3 mmol) in
DMF (2 mL) was added dropwise to the solution. The solution
was heated at 50 °C for 9 h and then cooled to room temperature
and concentrated. To the residue was added H2O (10 mL), and the
solution was neutralized with acetic acid. The solution was
concentrated and purified by silica gel column chromatography (90:
10:0.5 CHCl3/MeOH/NH4OH) (Rf 0.25). The collected product (605
mg) was suspended in acetic anhydride (20 mL) and heated at 150
°C. After 1 h, the solution was concentrated to dryness and to the
residue was added 20 mL of ammonium hydroxide (28% ammonia).
The solution was transferred to a sealed vessel and heated at 60 °C
for 1 h. The solution was concentrated to dryness and purified by
silica gel column chromatography (90:10:0.5 CHCl3/MeOH/
2.10. 6-Chloro-7-(2-(2,2-dimethoxyethoxy)ethyl)-7H-purin-2-
amine (10). 2-Amino-6-chloropurine (1.0 g, 5.9 mmol) was
suspended in DMF (15 mL) under a N2 atmosphere. 1,8-
Diazabicyclo[5.4.0]undec-7-ene (1.08 mL, 7.25 mmol) and 4 (1.34
g, 5.89 mmol) were added, and the solution was stirred at 80 °C
for 18 h. The solution was cooled to room temperature and
concentrated under reduced pressure. The residue was dissolved
in ethyl acetate (100 mL) and H2O (100 mL). The organic extract
was collected, and the aqueous extract was washed with ethyl
acetate (2× 100 mL). The organic extracts were combined, dried
over Na2SO4, filtered, concentrated, and purified by silica gel
column chromatography (95:5 CHCl3/EtOH) (Rf 0.15) to yield a
1
NH4OH) (Rf 0.15) to yield a white solid (50% yield). H NMR
(DMSO) δ 3.17 (6H, s), 3.36 (2H, d, J ) 5.0 Hz), 3.72 (2H, t, J
) 5.0 Hz), 4.31 (1H, t, J ) 5.5 Hz), 4.54 (2H, t, J ) 4.6 Hz), 6.87
(2H, s), 8.18 (2H, s).13C NMR (DMSO) δ 159.8, 152.0, 151.6,
146.4, 111.1, 102.1, 70.2, 69.99, 53.5, 46.3. λmax ) 270 nm (47, 54).
Anal. Calcd for C11H17N5O3: C, 49.43; H, 6.41; N, 26.20. Found:
C, 49.40; H, 6.48; N, 26.10.
2.6. N-(2-(2,2-Dimethoxyethoxy)ethyl)-9H-purin-6-amine (6).
6-Chloropurine (200 mg, 1.3 mmol) was dissolved in 2-(2,2-
dimethoxyethoxy)ethanamine (55) (∼1 mL, 5.2 mmol). The solution
was stirred at 70 °C for 2 h. The solution was cooled to room
temperature and concentrated to dryness. The resulting solid was
purified by silica gel column chromatography (85:15:0.5 CHCl3/
MeOH/TEA) (Rf 0.20) to yield a white solid that was recrystallized
from ethanol (95% yield).1H NMR (D2O) δ 3.40 (6H, s), 3.62 (2H,
d, J ) 5.5 Hz), 3.79-3.85 (4H, m), 4.59 (1H, t, J ) 5.0 Hz), 8.12
(1H, s), 8.20 (1H, s).13C NMR (D2O) δ 153.1, 151.7, 148.6, 139.9,
116.4, 102.7, 69.62, 69.48, 54.6, 40.2. Anal. Calcd for C11H17N5O3:
C, 49.43; H, 6.41; N, 26.20. Found: C, 49.44; H, 6.42; N, 26.06.
2.7. 3-(2-(2,2-Dimethoxyethoxy)ethyl)-3H-purin-6-amine (7). To
a solution of N-(4,6-diaminopyrimidin-5-yl)formamide (47) (1.0 g,
6.5 mmol) in dry DMF (75 mL) under N2 was added 4 Å molecular
sieves (4.8 g). The suspension was stirred and heated at 90 °C until
the amide was dissolved. To this solution was added 4 (2.23 g,
9.75 mmol), and this solution was heated at 90 °C for 11 days.
The resulting suspension was cooled to room temperature and
filtered. The filtrate was concentrated under vacuum to yield an oil
that was purified by silica gel column chromatography (90:10:0.5
CHCl3/MeOH/NH4OH) (Rf 0.29) yielding a white solid (7.5%).1H
NMR (CDCl3) δ 3.28 (6H, s), 3.41 (2H, d, J ) 5.5 Hz), 3.94 (2H,
t, J ) 5.0 Hz), 4.34 (1H, t, J ) 5.0 Hz), 4.54 (2H, t, J ) 4.6 Hz),
8.02 (1H, s), 8.1 (1H, s).13C NMR (CDCl3) δ 155.2, 153.7, 150.3,
143.6, 121.1, 102.6, 70.9, 68.5, 54.2, 50.2. λmax ) 272 nm (47, 54).
Mass Calcd for C11H18N5O3: 268.1406. Mass Obsd: 268.1404.
2.8. 9-((2R,4R,5R)-4-(tert-Butyldimethylsilyloxy)-5-((tert-bu-
tyldimethylsilyloxy)methyl)-tetrahydrofuran-2-yl)-6-(2-(2,2-dimethox-
yethoxy)ethoxy)-9H-purin-2-amine (8). The compound 2-amino-
9-((2R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-
butyldimethylsilyloxy)methyl)-tetrahydrofuran-2-yl)-1H-purin-6(9H)-
one (diTBDMS-dG) (56) (1.0 g, 2 mmol), triphenylphosphine (680
mg, 2.6 mmol), and 2-(2,2-dimethoxyethoxy)ethanol (400 mg, 2.6
mmol) were suspended in dry THF (40 mL) and stirred at 0 °C
1
white solid (15.6% yield). H NMR (CDCl3) δ 3.30 (6H, s), 3.42
(2H, d, J ) 5.0 Hz), 3.82 (2H, t, J ) 5.0 Hz), 4.35 (1H, t. J ) 5.0
Hz), 4.49 (2H, t, J ) 5.0), 5.06 (2H, s), 8.04 (1H, s).
2.11. 2-Amino-7-(2-(2,2-dimethoxyethoxy)ethyl)-1H-purin-6(7H)-
one (11). The compound 10 (100 mg, 0.33 mmol), K2CO3 (46 mg,
0.33 mmol), and 1,4-diazabicyclo[2.2.2]octane (46 mg, 0.41 mmol)
were suspended in water (7 mL) and heated in a sealed reaction
vial at 110 °C for 3 h. The solution was cooled to room temperature,
and the pH of the solution was adjusted to 7 using solid KH2PO4.
The precipitate that formed was filtered and dried under vacuum
to yield a white solid (95% yield). 1H NMR (DMSO) δ 3.21 (6H,
s), 3.37 (2H, d, J ) 5.5 Hz), 3.76 (2H, t, J ) 5.0 Hz), 4.30-4.36
(3H, m), 6.10 (2H, s), 7.85 (1H, s), 10.8 (1H, bs).13C NMR (DMSO)
δ 160.0, 154.9, 152.9, 143.6, 107.8, 102.2, 69.85, 69.45, 53.4, 45.8.
HRMS Calcd for C11H17N5O4: 284.1366. Obsd: 284.1353.
2.12. 2-(2-(2,2-Dimethoxyethoxy)ethylamino)-9-((2R,4R,5R)-4-
hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1H-purin-6(9H)-
one (12). The compound 9-((2R,4R,5R)-4-(tert-butyldimethylsilyloxy)-
5-((tert-butyldimethylsilyloxy)methyl)-tetrahydrofuran-2-yl)-2-fluoro-
6-(4-nitrophenethoxy)-9H-purine (57) (380 mg, 0.59 mmol) was
dissolved in 6 mL of methanolic 2-(2,2-dimethoxyethoxy)etha-
namine (1 M, 6.45 mmol) and stirred at room temperature for 18 h.
The solution was concentrated under reduced pressure. The residue
was dissolved in DMF (10 mL), and potassium fluoride (290 mg,
5.02 mmol) and 18-crown-6 (156 mg, 0.59 mmol) were added. The
suspension was heated at 60 °C for 18 h. The suspension was cooled
to room temperature and filtered. The filtrate was concentrated under
reduced pressure and purified as a single peak by HPLC using a
CH3CN/H2O gradient on a 5 µm C18 250 mm × 21.2 mm
semipreparative column (30% yield). Major form. (89%) 1H NMR
(DMSO) δ 2.20 (1H, m), 2.60 (1H, m), 3.27 (6H, s), 3.43-3.60
(8H, m), 3.80 (1H, m), 4.35 (1H, m), 4.46 (1H, t, J ) 5.0), 4.88

1226 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Zink et al.
(1H, t, J ) 5.5), 5.28 (1H, d, J ) 3.7), 6.15 (1H, t, J ) 6.0), 6.46
(1H, bs), 7.91 (1H, s), 10.6 (1H, s).13C NMR (DMSO) δ 156.7,
152.6, 150.40, 135.84, 116.93, 102.2, 87.6, 82.8, 70.89, 70.0, 69.1,
61.83, 53.4, 39.2. Minor form. (11%) 1H NMR (DMSO) δ
2.23-2.28 (1H, m), 2.69 (1H, m), 3.27 (6H, s), 3.43-3.60 (8H,
m), 4.08 (1H, m), 4.28 (1H, m), 4.46 (1H, t, J ) 5.0 Hz), 4.81
(1H, t, J ) 5.5 Hz), 5.49 (1H, d, J ) 4.1 Hz), 6.15 (1H, t, J ) 6.0
Hz), 6.46 (1H, bs), 8.01 (1H, s), 10.6 (1H, s).13C NMR (DMSO) δ
156.7, 152.6, 150.30, 136.3, 116.63, 102.2, 88.0, 82.8, 70.53, 70.0,
69.1, 61.55, 53.4, 39.2. HRMS Calcd for C16H26N5O7: 400.1836.
Obsd: 400.1827.
a buffered aqueous solution (0.05 M 70% anion cacodylic acid
buffer, pH 6.9) containing tris(2-carboxyethyl)phosphine hydro-
chloride (TCEP) and (15N6)-2′-deoxyadenosine or (15N2)-2′-deox-
yguanosine. The final concentrations were 10 mM in 3, 10 mM in
TCEP, and 0.5 mM in (15N6)-2′-deoxyadenosine or (15N2)-2′-
deoxyguanosine. The reaction was stirred at room temperature for
10 s with bubbling occurring. The solution was acidified to 0.1 M
HCl and subjected to mild acid hydrolysis (30 min, 80 °C). The
solution was cooled briefly, neutralized with NaOH (pH 7), and
diluted with synthetic (14N) EA standards. The products were
chromatographically separated and analyzed by LC/MS employing
single ion monitoring that monitored masses of 222.22 [M(14N)H+]
and 223.22 [M(15N)H+] for (2-ethoxyacetaldehyde)-adenine (EA-
Ade), 180.08 [M(14N)H+] and 181.08 [M(15N)H+] for (2-hydroxy-
ethoxy)-adenine (HE-Ade), 238.09 [M(14N)H+] and 239.09
[M(15N)H+] for (2-ethoxyacetaldehyde)-guanine (EA-Gua), and
196.08 [M(14N)H+] and 197.08 [M(15N)H+] for (2-hydroxyethyl)-
guanine (HE-Gua). The identity and the yields of products from
the reactions were determined from the peaks of mass [M(15N)H+],
which coeluted with the peaks of the standards [M(14N)H+]. Product
yields were calculated from the peak areas corrected for the
contribution of the M + 1 signal of the [M(14N)H+] standard. The
yields, standard deviations, and percent error were determined from
the averages of three experiments with the value from each
experiment being an average of at least three injections. Control
experiments involved the incubation of the purine nucleosides
in the decomposition products of 3 in aqueous buffer, followed
by acid hydrolysis, neutralization, separation, and analysis by
LC/MS.
2.13. 2-Amino-9-((2S,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-1-(2-(2,2-dimethoxyethoxy)ethyl)-1H-purin-
6(9H)-one (13). Guanosine hydrate (1.0 g, 3.53 mmol) and powdered
K2CO3 (0.73 g, 5.30 mmol) were suspended in DMF (25 mL). The
suspension was heated at 90 °C to solublize the guanosine. To the
solution was added 2-(2-bromoethoxy)-1,1-dimethoxyethane (58)
(1.13 g, 5.30 mmol), and the solution was heated at 90 °C for 24 h.
The solution was cooled to room temperature and filtered through
Celite. The filtrate was concentrated under reduced pressure and
purified by silica gel column chromatography (90:5:5 CH3CN/H2O/
1
NH4OH) (Rf 0.20) to yield a white solid (80% yield). H NMR
(DMSO) δ 3.24 (6H, s), 3.43 (2H, d, J ) 5.0 Hz), 3.50-3.65 (4H,
m), 3.86 (1H, q), 4.09 (1H, t, J ) 4.5 Hz), 4.14 (2H, t, J ) 5.5 Hz
Hz), 4.41 (2H, t, J ) 5.5), 5.02 (1H, bs), 5.13 (1H, bs), 5.40 (1H,
bs), 5.70 (1H, d, J ) 6.0 Hz), 6.93 (2H, s), 7.95 (1H, s).13C NMR
(DMSO) δ 156.5, 154.3, 149.5, 135.9, 116.0, 102.2, 86.0, 85.2,
73.6, 70.4, 70.1, 68.2, 61.4, 53.5, 40.8. HRMS Calcd for
C16H26N5O8: 416.1780. Obsd: 416.1776.
2.16.2. Alkylation of DNA. These experiments were initiated
when a solution of 3 (Scheme 1) in acetonitrile was added to a
buffered aqueous solution (0.05 M 70% anion cacodylic acid buffer,
pH 6.9) containing tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) and calf thymus DNA. The final concentrations were 10
mM in 3, 10 mM TCEP, and ∼0.5 mg/mL calf thymus DNA. The
reaction was stirred at room temperature for 15 s with bubbling
occurring. The solution was acidified to 0.1 M HCl and subjected
to mild acid hydrolysis (30 min, 80 °C). The solution was cooled
briefly, neutralized with NaOH (pH 7), and separated into three
aliquots. Two of the aliquots were chromatographically separated
and analyzed by LC/MS employing single ion monitoring for the
analysis of EA-Ade (MH+ ) 222.22), HE-Ade (MH+ ) 180.08),
EA-Gua (MH+ ) 238.09), and HE-Gua (MH+ ) 196.08) products.
The third aliquot was chromatographically separated and analyzed
by HPLC employing a UV/vis PDA detector to determine the
concentrations of Ade and Gua in the calf thymus DNA.
2.14. 2-(2,3-Dihydro-1,4-oxazin-4-yl)-1-H-purin-6(9H)-one (14).
To 25 mg of 12 was added 5 mL of a solution of D2O containing
0.1 M DCl, and this was stirred at 80 °C for 30 min. Analysis by
HPLC, employing a 10 mM Ammonium Acetate pH 6.9/CH3CN
gradient at a flow rate of 1 mL/min and a C18(2) 250 × 4.6 mm
column, indicated a minor component of fused peaks at 38-39
min followed by the major peak at 49 min, the balance of which
was isolated by use of a semipreparative column. The eluate was
concentrated in vaccuo, but not to dryness, and to this concentrated
fraction was added 5 volume equivalents of D2O. This procedure
was repeated 5 times subsequently, the last two times employing
99.996 atom % D, D2O, after which the product was analyzed
spectroscopically. 1H NMR (DMSO) δ 3.85 (2H, t, J ) 4.6 Hz),4.11
(2H, t, J ) 4.9 Hz), 6.17 (1H, d, J ) 5.0 Hz), 6.59 (1H, d, J ) 5.0
Hz), 8.27 (1H, s), 11.45 (1H, bs).13C NMR (DMSO) δ 155.8, 151.8,
148.2, 137.8, 130.1, 118.1, 112.1, 105.5, 64.3, 39.9. [MH+] )
220.2.
2.15. Generation and Characterization of EA-Nucleobase Stan-
dards. The dimethoxy-protected nucleobases or nucleosides 5, 6,
7, 9, 11, 12, and 13 were subjected to mild acid hydrolysis to
liberate their corresponding aldehydes. They were subsequently
employed as standards in later experiments carried out to character-
ize nucleoside and DNA alkylation by the diazonium ion derived
from R-hydroxynitrosomorpholine. Deprotection was carried out
by preparing a solution of the nucleobase/nucleoside in D2O
(99.996% D) and adding DCl to yield a final concentration of ∼7
mM nucleobase/nucleoside and 0.1 M DCl. The reaction solution
was maintained at 25 °C (nucleobase) or 80 °C (nucleoside). The
The products of the reaction were identified using authentic
standards with the yields being calculated from standard curves.
Product yields, standard deviations, and percent error were deter-
mined from the averages of three experiments with the value from
each experiment being an average of at least three injections.
Control experiments involved the incubation of 2, generated from
the reduction of 3, in aqueous buffer at 23 °C for 15 s. Calf thymus
DNA was then incubated in the reaction solution containing the
decomposition products of 2 for 10 s, followed by acid hydrolysis,
neutralization, separation, and analysis by LC/MS. No EA-
nucleobase products were detected in these control experiments.
2.17. Kinetics. 2.17.1. General. All rate constants were de-
termined under initial rate conditions monitoring less than 10% of
conversion. Kinetics of decay of the ethoxyacetaldehyde (EA)
nucleobase adducts were determined by monitoring the disappear-
ance of the starting material monitored with a photodiode array
detector and by comparison with the internal standard 5-benzimi-
dazolecarboxylic acid. Rates of formation of hydroxyethyl (HE)
adducts from EA adducts of nucleoside or DNA that were formed
in the reactions of R-hydroxynitrosomorpholine were determined
by monitoring the appearance of the HE adducts with time by LC/
MS. Rates of depurination of EA adducts from DNA were
determined by monitoring the appearance and EA-nucleobase
adduct as a function of time by LC/MS using the internal standard
5-benzimidazolecarboxylic acid.
1
reactions were monitored by H NMR following the decay of the
dimethoxy methine signal (∼4.50 ppm) and the decay of the H1′
signal from the ribose sugar (∼6.40 ppm) (nucleoside products).
With two exceptions (see Results), the resulting aldehyde is seen
predominantly in its hydrate form with the new methine signal
forming around 5.05 ppm. Dimethylformamide was used as an
internal standard to quantify the product yields. After completion
of the reaction and characterization, the resulting solutions were
stored at -20 °C and were used in experiments as stock solutions
for authentic standards. Periodic checks indicated that the products
were stable to the storage conditions.
2.16. Products of Alkylation by r-Hydroxynitrosomorpho-
line. 2.16.1. Alkylation of Nucleosides. These experiments were
initiated when a solution of 3 (Scheme 1) in CH3CN was added to

Direct Reaction of the Diazonium Ion with Purines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1227
Scheme 2. Precusors Synthesized for the Detection of Some
Prospective Purine Adducts of the Diazonium Ion from
N-Nitroso-3-hydroxy-morpholine
Table 1. Removal of the Dimethoxy Protecting Group and
the Ribose Sugar from Nucleobase and Nucleoside Standards
Using 0.1 M DCl in D2O and 7.5 mM DMF (Internal
Standard)
nucleosidea
temperature
dimethoxy removal
sugar cleavage
5
6
7
9
R.T.
R.T.
R.T.
R.T.
80 °C
R.T.
80 °C
R.T.
92%/48 h
98%/24 hb
98%/48 h
95%/48 h
95%/30 min
91%/44 h
96%/30 min
100%/33 hc
100%/20 mind
100%/30 min
95%/15 min
100%/15 min
11
12
13
95%/19 h
100%/20 min
90%/5 h
80 °C
80 °C
2.17.2. Examination of the Aqueous Stability of 2-(2-Ethoxy-
acetaldehyde)-Nucleobase Standards. The decay of the (2-ethoxy-
acetaldehyde)-nucleobase (EA-nucleobase) standards was studied
in aqueous buffered media at 25 °C using a Waters 717 HPLC
with an autosampler, a temperature controlled carousel, and a
Waters 996 photodiode array detector monitoring at 270 nm.
Separations were performed on a Restek Pinnacle II reverse phase
C18 5 µm, 250 × 4.6 mm column and effected by means of a
H2O/CH3CN gradient. The buffers used in these experiments were
carbonate, phosphate, and ethanolamine. Reaction solutions were
maintained at ionic strength µ ) 1.0 M, using KCl. Experiments
were initiated when a solution containing the EA-nucleobase
dissolved in water was injected into the reaction vial and then
injected onto the HPLC. The final concentration of the EA-
nucleobase was (1-2) × 10-4 M.
a Concentration is 6-7 mM. b 77% diol, 22% carbinolamine, and 1%
aldehyde forms. c 33% diol, 45% carbinolamine, and 22% alkene forms.
d 12% diol, 16% carbinolamine, and 72% alkene forms.
(nucleosides) to liberate the corresponding aldehydes and to
generate the free nucleobases, respectively, as in eq 2.
The reactions were monitored by following the decay of the
dimethoxy methine signal (∼4.50 ppm) and the decay, where
applicable, of the H1′ signal from the ribose sugar (∼6.40 ppm).
The corresponding aldehyde under these conditions is in its
hydrate form (>98%) with the new methine hydrogen signal
appearing around 5.05 ppm. The conversion times and percent
yields, based on the comparison with an internal standard DMF,
for these reactions are illustrated in Table 1. Injection, after
neutralization of a portion of the reaction solution, on HPLC
with UV/vis detection resulted in a single UV absorbing peak
with an absorption spectrum consistent with what has been
reported for simple alkyl group substitution at the site expected.
Use of ESI/MS detection again yielded a peak with a spectrum
consistent with the hydrate form. In some cases, hydrolysis also
resulted in conversion to the hydroxyethylated nucleobase, to
the extent of 1-3%.
2.17.3. Formation of Hydroxyethylated Nucleobases. Experi-
ments monitoring the rates of formation of HE adducts from
nucleosides or DNA reacted with R-hydroxynitrosomorpholine were
initiated when a solution of 3 in acetonitrile was added to a buffered
aqueous solution (0.05 M 70% anion cacodylic acid buffer, pH
6.9) containing tris(2-carboxyethyl)phosphine hydrochloride (TCEP)
and nucleoside (0.5 mM) or calf thymus DNA (0.5 mg/mL). The
final concentrations of 3 and TCEP were 10 mM. The reaction was
stirred at room temperature for 15 s, with evolution of gas noted
during this period. Aliquots of the reaction were removed periodi-
cally and subjected to acid hydrolysis by neutralization of the buffer
base and addition of HCl from a concentrated stock to give a
concentration equal to 0.1M, after which the sample was hydrolyzed
at 80 °C for 30 min. The products were separated and analyzed by
LC/MS. Single ion monitoring was employed to analyze for masses
180.08 (HE-Ade) and 196.08 (HE-Gua) over the course of the
reaction.
2.17.4. Rates of Depurination of Duplex DNA Subsequent
to Reaction with r-Hydroxynitrosomorpholine. Experiments
monitoring the rates of depurination of DNA reacted with R-hy-
droxynitrosomorpholine were initiated when a solution of 3 in
acetonitrile was added to a buffered aqueous solution (0.05 M 70%
anion cacodylic acid buffer, pH 6.9) containing TCEP and calf
thymus DNA. The final concentrations were 10 mM in 3, 10 mM
TCEP, and ∼0.5 mg/mL calf thymus DNA. The reaction was stirred
at room temperature for 15 s, with evolution of gas noted during
this period. Aliquots of the reaction were removed periodically,
and products were chromatographically separated and analyzed by
LC/MS without acid induced hydrolysis. Single ion monitoring was
employed to analyze for masses 222.22 (EA-Ade) and 238.09 (EA-
Gua) over the course of the reaction.
3.1.2. Hydrolysis of 6. Monitoring of the 1HNMR spectrum
of the decay of 6 in 0.1 M DCl in D2O at room temperature for
24 h gave evidence of the complete conversion, on the basis of
the internal standard DMF, of the methinyl hydrogen to three
different forms consistent with chemical shifts expected of the
aldehyde, 9.65 ppm (∼1%), the aldehyde hydrate, 5.05 ppm
(77%), and a carbinolamine, 6.56 ppm (22%). Analysis by
HPLC with diode array detection exhibited a single UV-
absorbing peak, while ESI/MS detection similarly indicates a
single peak containing signals with MH+ consistent with all
three structures. Neutralization to pD ) 5.1 and reanalysis by
1HNMR indicates a shift in the equilibrium of the three species
with the carbinolamine form becoming dominant at 83%, the
aldehyde hydrate form being 16%, and the aldehyde remaining
1
at ∼1%. Analysis of the H-13C coupling of this sample by
HMBC (Figure 1) indicates a structure consistent with 15 on
the basis of coupling between H3′ (6.56 ppm) and both C5′
(40.1 ppm) and C6 (153.1 ppm).
3. Results
3.1. Generation and Characterization of EA-Nucleobase
Standards from Dimethoxyprotected Nucleobases and Nucleo-
sides. Structures of the relevant compounds are summarized in
Scheme 2.
3.1.1. Hydrolysis of 5, 7, 9, 11, and 13. The dimethoxy-
protected compounds were subjected to mild acid hydrolysis
using 0.1 M DCl in D2O at 20 ( 2 °C (nucleobases) or 80 °C
3.1.3. Hydrolysis of 12. Monitoring of the 1HNMR
spectrum of the decay of 12 in 0.1 M DCl in D2O at room
temperature 20 ( 2 or 80 °C for 30 min indicates the complete
disappearance of the methinyl H of 12 to yield three different
forms consistent with chemical shifts expected of the aldehyde,
9.65 ppm, the aldehyde hydrate, 5.05 ppm, and a carbinolamine,

1228 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Zink et al.
Figure 1. HMBC spectrum of the acid hydrolysis product 15 of
compound 6. Key long-range couplings are circled.
Figure 2. HMBC spectrum of the acid hydrolysis product 14 of
compound 12. Key long-range couplings are circled.
pholine, 3, was decomposed by the addition of TCEP to
solutions of 15N6-dAde, 15N2-dGua, or DNA, and the EA-
containing nucleobases, after acid hydrolysis, were separated,
detected, and quantified by HPLC-MS facilitated by selected
ion monitoring. In the case of the nucleoside reactions,
identification and quantitation were effected by the addition of
natural abundance isotope-containing adduct standards after
deprotection (as described above). In the case of DNA,
identification was confirmed by coelution under two different
chromatography conditions and spiking with authentic standards,
and quantitation was effected by interpolation from three point
standard curves. Typical chromatograms, for the alkylation of
dAde and DNA, are presented in Figures 4 and 5, respectively.
In the reactions with DNA, three adducts, 15, N1-EA-Gua, and
N2-Gua adducts in the form of either N2-EA-Gua (hydrate) or
compound 14, were undetected using a normal 10 µL sample
injection. A 10-fold larger injection volume revealed a quantifi-
able amount of the last of these: N2-EA-Gua (hydrate). A
summary of the percentage yield of each of the adducts detected
is include in Table 3.
6.56 ppm. The aldehyde is in trace quantities, <1%, under both
hydrolysis conditions. In addition, under both conditions, two
doublets in the alkene region, 6-7 ppm region, appear. Isolation
and characterization of the compound responsible for these
resonances was effected by chromatography. The HMBC
spectrum (Figure 2) indicates the H3′ f C5′, H3′ f C2, and
H5′ f C2 couplings consistent with the structure of compound
14. The alkene predominates, at the expense of the aldehyde,
aldehyde hydrate, and carbinolamine in the higher temperature
hydrolysis. For hydrolysis at room temperature, the ratio of
1
hydrated aldehyde/carbinolamine/alkene, calculated from H
NMR, is 33/45/22, but changes to 12/16/72 when the temper-
ature of hydrolysis is raised to 80 °C.
The hydrolysis time required and the conversion yields for
the deprotection and or deglycosylation to yield the EA-
nucleobase standards are summarized in Table 1.
3.3. Transformation of EA Adducts of Nucleosides and
DNA Reacted with r-Hydroxynitrosomorpholine. 3.3.1. For-
mation of HE Purine Adducts. The conversion of the EA-Ade
and EA-Gua products, which were generated in the reactions
of 2, from 3, in the presence of nucleosides or DNA, to HE
adducts was examined by monitoring the formation of HE
adducts as a function of time. In these reactions, 3 was reduced
with TCEP in a buffered solution containing dAde, dGua, or
DNA. The solution was stirred at room temperature, and aliquots
were removed periodically. The aliquots were hydrolyzed in
acid and neutralized, and the products were chromatographically
separated and analyzed by LC/MS. Increases in the formation
of HE adducts as a function of time were plotted as illustrated
in Figure 3 for the N7-HE-Gua and O6-HE-Gua adducts of dGua
(panel B) and all four of the major adducts observed in DNA
(panel C). Slopes of such plots allowed the calculation of the
values of kobsd, which are summarized in Table 4.
3.3.2. Spontaneous Depurination of EA Adducts from
DNA Reacted with r-Hydroxynitrosomorpholine. R-Hydro-
peroxy-N-nitrosomorpholine, 3, was reduced by TCEP in the
presence of calf thymus DNA. As a function of time, aliquots
of the reaction solution were removed and subjected to LC
separation, without prior acid hydrolysis, and free nucleobase
EA adducts were detected by MS. Typical plots of the increases
in liberated nucleobase EA adducts as a function of time are
indicated for the adenine adducts in Figure 3, panel D. Rate
3.1.4. Stability of Nucleobase Standards in Neutral and
Basic Media: Decay of EA Adducts. The rate of decomposition
of nucleobase standards was monitored under various conditions
including phosphate and carbonate buffered media, in some
cases containing ethanolamine. Typical data for the initial rates
of decay of Gua adducts in carbonate buffer, pH 10.4, monitored
by UV detection and employing the internal standard 5-benz-
imidazole carboxylic acid, are shown in Figure 3, panel A. It
was not possible to accurately measure rate constants for decay
at neutral pH buffered with phosphate without the addition of
ethanolamine, which considerably elevated the rate of decay.
Calculated rate constants for the disappearance of the EA
adducts under different conditions are summarized in Table 2.
Values of kobsd for the EA adduct attached to the exocyclic amino
group of Gua, N2-EA-Gua, could not be measured due to
irregularities in peak shape possibly due to the equilibrium
between the hydrated aldehyde/aldehyde/alkene forms that could
not be separated. Finally, for comparison, the rate constants were
also measured for the EA adduct of benzimidazole, whose
chemistry has been previously characterized, and these values
are also included in Table 2 (41).
3.2. Alkylation of Purine Nucleosides and Purines in DNA
by r-Hydroxynitrosomorpholine. R-Hydroperoxynitrosomor-

Direct Reaction of the Diazonium Ion with Purines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1229
Figure 3. Kinetics of decay or formation of adducts on nucleobases or DNA. (A) Decay of EA-Gua adducts, N1-EA-Gua (9), N7-EA-Gua (b), and
O6-EA-Gua (() as a function of time at 25 °C, pH 10.4, and 0.01 M carbonate buffer. (B) Formation of N7-HE-Gua (9) and O6-HE-Gua (b)
adducts as a function of time at 25 °C, 0.05 M 70% anion cacodylic acid buffer, and pH 6.8. (C) Formation of O6-HE-Gua ((), N7-HE-Gua (9),
N3-HE-Ade (2), and N7-HE-Ade (b) adducts from the respective EA-DNA adducts as a function of time at 25 °C, 0.05 M 70% anion cacodylic
acid buffer, and pH 6.9. (D) Spontaneous depurination to form EA-Ade nucleobase adducts N3-EA-Ade (9) and N7-EA-Ade (b) from reactions
with DNA at 25 °C, 0.05 M 70% anion cacodylic acid buffer, and pH 6.9.
Table 2. Rate Constants for the Decay of EA-Nucleobase
Standards and EA-Benzimidazole (bzi) in Aqueous Buffered
Solutions (0.1 M) at 25 °C and 1 M Ionic Strength (KCl)
107 × kobsd (s-1
)
phosphate
pH 7.4 0.1 M
ethanolamine
carbonate
pH 10.4
carbonate
pH 9.6
phosphate
pH 7.4
bzi
17.6
15.5
11.2
11.8
6.52
4.39
5.32
4.91
8.18
6.74
3.75
2.71
<1.24
2.54
<0.02
<1.59
<0.07
<0.17
<0.17
<0.30
<0.38
63.6
37.3
<1.1
36.0
18.9
48.8
51.5
N3-Ade
N6-Ade
N7-Ade
N1-Gua
O6-Gua
N7-Gua
constants based on the initial rates of the reaction were calculated
assuming an end point of complete depurination, on the basis
of the initial yield of the EA adduct calculated from acid
hydrolysis of an aliquot after the initial alkylation reaction. The
rate constants and calculated half-times for the relevant modified
nucleobases are summarized in Table 5.
Figure 4. Typical LC/MS chromatogram with single ion monitoring
for the reaction of 3, reduced with TCEP, with (15N6)-2′-deoxyadenosine,
25 °C, 0.05 M 70% anion cacodylic acid buffer, and pH 6.9 and analysis
for EA-Ade adducts. (a) m/z ) 241 for N3 and N7 (15N6)-EA-Ade
(hydrate forms). (b) m/z ) 240 for N3 and N7 (14N6)-EA-Ade standards
(hydrate forms). (c) m/z ) 223 for N3 and N7 (15N6)-EA-Ade (aldehyde
form) and (15N6)-15. (d) m/z ) 222 for N3 and N7 (14N6)-EA-Ade
(aldehyde form) and (14N6)-15. The peaks marked with an asterisk were
identified in the control experiments.
4. Discussion
4.1. Dimethylacetal Precursors to Ethoxyacetaldehyde
Adducts of Purine Nucleobases. The use of dimethylacetals
to generate dilute solutions of nucleobases containing the
ethoxyacetaldehyde moiety was adopted on the basis of
preliminary work in which attempts to make pure nucleobase
adducts containing the pendant aldehyde met with failure due
to oligomer- and polymerization and other instabilities manifest
by the aldehyde functionality. Generally, under the influence
of acid and in some cases heat for deglycosylation, the dilute
solutions of acetals were converted to predominantly the
aldehyde hydrate form in nearly quantitative yield (Table 1).
Two exceptions were the EA adducts attached to the exocyclic
amino groups of the purines. Cleavage of the dilute acetals 6

1230 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Zink et al.
defined peak in HPLC/MS that could be used in further adduct
quantitation and analysis.
The EA-nucleobase adduct liberated by acid hydrolysis from
the acetals was not markedly destabilized by attachment to the
various heteroatoms of the purines. While decomposition to the
HE-nucleobase was detected by mass spectrometry to the extent
of 1-3% during acid hydrolysis of the dimethylacetals, the
kinetics of decay studied at neutral and basic pH indicated that
the stability of the EA moiety attached to the purines is
comparable to that when attached to benzimidazole. In an earlier
study, it was noted that the EA-fragment attached to benzimi-
dazole was quite stable at neutral pH, but its decay was
accelerated in basic media and in the presence of primary and
secondary amines (41). Thus, the possibility that the pendant
amines of the purines might destabilize the EA fragment was
anticipated. To the contrary, examination of the data in Table
2 indicates, in general, a very similar stability, with rate constants
for decay generally within a factor of 3 of that observed with
the benzimidazole adduct. In no case was it possible to reliably
determine the rate constants for decay in nearly neutral
phosphate buffer (Table 2) due to the sluggishness of the
reaction. As previously noted with the benzimidazole adduct,
ethanolamine accelerates the decay, except in the case of the
N6-EA-Ade adduct. The reason for enhanced stability of the
N6-EA-Ade adduct is unclear; it may have to do with adoption
of the carbinolamine form, 15, though this does not appear to
imbue stability in basic pH, to the extent that it exists as such
under those conditions. Stability of the N2-EA-Gua adduct could
not be reliably monitored by initial rate methods due to the poor
chromatographic resolution that resulted in excessive scatter in
the initial rate plots of concentration against time.
4.2. Deposition of EA Adducts in the Decay of r-Hydrox-
ynitrosomorpholine in the Presence of Nucleosides and Duplex
DNA. 4.2.1. Nucleosides. Consistent with what is expected on
the basis of alkylation by simple primary diazonium ions, all
sites investigated are targeted by the diazonium ion intermediate
and, similarly, with a distinct preference for the N7 and O6 atoms
of dGua. The 1-propyldiazonium ion similarly targets, for
n-propylation, all heteroatoms on the purine nucleosides, with
the O6 and N7 of dGua being an average of 7-fold more
abundant than the third-most abundant adduct studied here, N7
of dAde (59). Interestingly, the range of yields is an order of
magnitude larger in the case of the diazonium ion derived from
R-hydroxynitrosomorpholine compared to what is observed in
n-propylation by the 1-propyldiazonium ion. The reason for this
is presently unclear; possibly, it is due to methodological
differences. In any case, there is no reason to expect quantitative
uniformity given that the diazonium ion in the present case is
sterically larger and can also undergo intramolecular capture,
both of which factors can impact product yields differently
depending upon the structure of the encounter complex with
the different atom sites on the nucleosides.
Figure 5. Typical LC/MS chromatogram with single ion monitoring
for the reaction of 3, reduced with TCEP, with calf thymus DNA, 25
°C, 0.05 M 70% anion cacodylic acid buffer, and pH 6.9, and analyzing
for EA-Ade and EA-Gua. (a) m/z ) 240 for N3 and N7-EA-Ade
(hydrate forms). (b) m/z ) 222 for N3 and N7-EA-Ade (aldehyde forms)
and 15. (c) m/z ) 256 for N7- and O6-EA-Gua (hydrate form). (d) m/z
) 238 for N7-, O6-, and N2-EA-Gua (aldehyde form). N2-EA-Gua is
not observed in this chromatogram (see Results). The peak marked
with an asterisk (*) was identified in the control experiments.
Table 3. Percent Yields of EA Products in Reactions of
Purine Nucleosides and DNA with HOONMOR in Aqueous
Media at 23 °C and pH 6.8
nucleoside reactions
DNA reactions
nucleobase
adduct
102 × average
102 × average
% yield
% error
% yield
% error
4.7
N3-EA-Ade
N6-EA-Ade
N7-EA-Ade
N1-EA-Gua
N2-EA-Gua
O6-EA-Gua
N7-EA-Gua
0.06
0.04
0.37
0.34
0.11
4.92
8.65
3.2
10.6
3.0
4.7
9.7
1.4
1.6
6.14
<0.035
1.52
<0.005
0.15
38.3
2.4
2.8
0.8
2.5
41.1
Table 4. Rate Constants of HE-Ade and HE-Gua Formation
in Aqueous Buffered Media at 23 °C
106 × kobsd (s-1
nucleosides
)
DNA
0.1 M phosphate 0.1 M cacodylate 0.1 M cacodylate
nucleobase adduct
(pH 7.4)
(pH 6.8)
(pH 6.8)
N3-HE-Ade
N7-HE-Ade
N7-HE-Gua
O6-HE-Gua
0.10
0.04
0.01
0.06
1.20
0.62
0.06
0.02
1.16
4.51
0.81
1.08
Table 5. Rates of Depurination from the DNA of EA
Adducts at pH 6.8, 23 °C and of Methylated (Me) Adducts
at pH 7.2, 37 °C
nucleobase
adduct
t1/2 (h) refs 61 and 62
nucleobase 106 × kobsd
106 × kobsd
adduct
(s-1
)
(s-1
)
t1/2 (h)
4.2.2. Duplex DNA. The O6 and N7 atoms of Gua are the
predominant sites of deposition in duplex DNA with N3 of Ade
being the third most targeted. This is qualitatively what is
observed with the ethyldiazonium ion and n-propylation by
1-propyldiazonium ions (59). Remarkably, EA deposition at the
N3 atom of Ade in the duplex increases 100-fold over what is
observed in the nucleoside, substantially larger than the 8-fold
increase in n-propylation at N3 observed in the case of the
1-propyldiazonium ion. The disappearance of adducts at N1 of
Gua and N6 of Ade in going from the nucleoside to the DNA
duplex was also noted in the earlier propylation study. Similarly,
the N2-EA-Gua remains a minor product in reactions of duplex
N3-EA-Ade
N7-EA-Ade
N7-EA-Gua
5.94
47.9
1.29
32.4
4.0
150
N3-Me-Ade
N7-Me-Ade
N7-Me-Gua
6.42
68.7
1.34
30
2.8
144
and 12, gave rise to cyclic structures 15 and 14, respectively.
The alkene-containing 14 predominated when the cleavage was
accelerated by heat and was used for quantifying adduct levels
(vide infra). Heating of 15 at 80 °C initiated the formation of
1
some H NMR resonances in the alkene region, but this was
not investigated further. The formation at room temperature of
15 and the hydrate and aldehyde forms that are in equilibrium
with it were essentially quantitative and gave a single well-

Direct Reaction of the Diazonium Ion with Purines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1231
DNA. Though minor, its biological significance is unknown.
Certainly, considering the importance of lesion persistence, it
is the only one of the adducts presently observed that will not
either spontaneously depurinate or for which there is no specific
repair mechanism such as the alkyl guanyl transferases in the
case of O6-Gua adducts. The activity of this repair protein is
presently undocumented for the lesions derived from R-hydrox-
ynitrosomorpholine, but the activity is known to be inhibited
and accelerated, relative to the removal of a simple methyl
group, by factors that similarly affect simple SN2 reactions.
Increased steric bulk and electron withdrawal decelerate rates
of repair. Conjugation adjacent to the reactive center accelerates
repair. The predominant form of the EA adduct, the hydrate,
contains both an electron withdrawing group and increased steric
bulk relative to a simple methyl group, suggesting the activity
of alkylguanyl transferase might be anticipated to be relatively
reduced compared to that of methyl. However, it is notable that
the protein retains substantial activity against the pyridyloxobutyl
lesion (60).
4.2.3. Comparison with Other Cyclic r-Hydroxynitro-
samines. Although a precise quantitative comparison is not
possible, the extant literature indicates that the diazonium ion
derived from R-hydroxynitrosomorpholine is of efficiency with
respect to alkylation of the N7 atom of Gua in the nucleoside
and DNA that is comparable to that of R-hydroxynitrosopyr-
rolidine. The diazonium ions from R-hydroxyNMOR and
R-hydroxynitrosopyrrolidine generated the adducts 16 and 17,
respectively. The alcohol derived from borohydride reduction
of 17 was the species actually detected. The N7 adduct, 17, is
the major product of alkylation of dGua by the precursor
R-acetoxynitrosopyrrolidine, with a yield of ∼8 mmol of adduct
per mol of Gua (31).
has also been reported. In some part, the difference may lie in
the differences in approach taken here and previously. The
present study has focused on diazonium ion adducts, using
synthetic standards and chromatographic methods to quantify
the product yields. By their nature, the diazonium ions are
relatively unselective and short-lived. In the present study for
the reactions with DNA, only ∼1% of the diazonium ions are
trapped by DNA. The overwhelming portion reacts with water
or by internal capture by the carbonyl of the diazonium ion
and yields much more stable aldehyde equivalents, with some
smaller fraction undergoing diazonium rearrangement and
carbocation hydration, yielding other, again, more stable car-
bonyl electrophiles. The earlier studies, with R-acetoxynitros-
opiperidine, are carried out with much longer exposure times
in which these more stable carbonyl electrophiles can modify
DNA, possibly substantially transforming the initial product
profile so that any initially deposited diazonium ion derived
products become relatively minor. The present study, using
R-hydroperoxynitrosomorpholine, generates the R-hydroxyni-
trosomorpholine in seconds, thereby generating the diazonium
ion in seconds (29), and after a total reaction time of only 15 s,
the products are subjected to acid hydrolysis, chromatographic
resolution, and detection. We stress that it is currently unknown
which adducts from which electrophiles engender the potent
mutagenic effects of any of the cyclic nitrosamines.
4.3. Formation of HE Adducts on Nucleosides and DNA.
Rate constants for the formation of HE adducts are in some
cases markedly accelerated in duplex DNA. Rate constants for
HE formation were determined for a limited number of EA
adducts, those that predominated in deposition on DNA.
Decomposition of R-hydroxynitrosomorpholine in the presence
of either nucleosides or DNA yielded the EA adducts, and the
subsequent formation of HE adducts was monitored as a function
of time. Comparison of the two rightmost columns in Table 4
indicate that the incorporation of the EA adduct into duplex
DNA accelerates the formation of the HE adduct in the cases
of the N7-EA-Ade, N7-EA-Gua, and O6-EA-Gua adducts by
factors of 7, 14, and 54, respectively. The basis for these rate
accelerations and their differences is currently uncertain. There
is no detectable acceleration of the formation of the HE adduct
upon incorporation into the DNA duplex in the case of the N3-
EA-Ade adduct. Of the four adducts studied, this is the only
one that lies in the minor groove, although at this point, it is
unknown if this is any more than coincidence.
Upon normalizing for the differences in the amounts of
alkylating agents to which the DNA was subjected in that and
the present study, the diazonium ion from R-hydroxynitroso-
morpholine is 3-fold less effective. With DNA as a target, 17
is the third most abundant product, but the most abundant
diazonium ion adduct with a yield of 6.6 mmol of adduct per
mol of Gua (32). Again correcting for the differences in amounts
of alkylating agent in the two studies, the diazonium ion derived
from R-hydroxynitrosomorpholine is 6-fold more efficient than
that derived from R-hydroxynitrosopyrrolidine. Methodological
differences between the present study and those of the Hecht
group are such that there is doubt about the precision of these
comparisons. For now, it is sufficient to suggest a similar
efficiency in the DNA alkylation by these diazonium ions until
such time as a direct comparison can be made using identical
precursors and reaction conditions.
The present results appear to contrast markedly with the
damage profile so far elucidated for the congener of R-hydrox-
ynitrosomorpholine, R-hydroxynitrosopiperidine. While the
alkylation profile by the former is typical of simpler diazonium
ions, not a single diazonium ion derived product has been
reported for the latter (30-32, 39). The major product from a
reaction of R-acetoxynitrosopiperidine involves the reaction of
the exocyclic amino group of dGua with the aldehyde equivalent
derived from hydrolysis of the diazonium ion. An etheno adduct
4.4. Kinetics of Depurination of EA Adducts. Rate constants
derived from initial rate measurements of DNA treated with
R-hydroxynitrosomorpholine presented in the left half of Table
5 suggest that these adducts are unremarkable in their reactivity.
The order of reactivity, N7-EA-Ade > N3-EA-Ade > N7-EA-
Gua, is identical with what has been observed for the analo-
gously methylated bases in DNA, as indicated by the data in
the right half of Table 5 (61, 62). These data are for the reaction
of the methylated bases at 37 °C, while the data for the EA
adducts are at 22 °C. A literature compilation of values for N7-
Me-Gua depurination suggests one might anticipate an increase
in rate constant by about a factor of ∼2 at 37 °C (61, 62). This
would make the purine EA adducts somewhat more reactive
than the methylpurines, but well within the range of values
observed for the depurination of N7-alkyl-Gua residues contain-
ing an electron withdrawing group a few atoms proximal to
the N7 atom.
4.5. Summary of Most Significant Findings. The first purine
adduct profile of the diazonium ion derived from R-hydroxyni-
trosomorpholine demonstrates remarkable similarity to those

1232 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Zink et al.
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observed for the diazonium ions derived from simple acyclic
R-hydroxynitrosamines. It contrasts for the most part with the
damage profiles reported to date for structurally related R-sub-
stituted cyclic nitrosamines. The ethoxyacetaldehyde moiety
initially deposited is relatively stable but slowly decays to a
hydroxyethyl fragment, and this decay is accelerated in duplex
DNA compared to that in nucleosides.
Acknowledgment. Work in this report was supported by the
National Cancer Institute, National Institutes of Health under
R01 CA52881.
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