Synthesis, characterization, and conformational analysis of DNA adducts from methylated anilines present in tobacco smoke

Article abstract of DOI:10.1021/tx950044z

The ability of a series of aromatic amines present in tobacco smoke (2-, 3-, and 4-methylaniline, 2,3- and 2,4-dimethylaniline) to bind to DNA has been investigated by reacting N-(acyloxy)arylamines with dG, dG nucleotides, and DNA. The predominant products from reactions with dG and the nucleotides were characterized as N-(deoxyguanosin-8-yl)arylamines by spectroscopic and HPLC methods. HPLC and spectroscopic analyses of the modified DNA indicated the same adducts. Analyses of the ¹H and ¹³C NMR spectra suggested that the adducts containing a methyl substituent ortho to the arylamine nitrogen had a higher percentage of syn conformers. This observation was supported by theoretical simulation studies that indicated substantial percentages of low energy syn conformers, increasing with the substitution pattern in the order para < meta < ortho < ortho,para < ortho,meta. The results demonstrate that, although single-ring arylamines are considered weak carcinogens, their electrophilic N-acetoxy derivatives, which are plausible metabolic intermediates, react with DNA to yield covalent adducts structurally identical to those derived from carcinogenic polyarylamines, such as 2- aminofluorene and 4-aminobiphenyl. Furthermore, the conformational perturbation induced in DNA by the formation of the monoarylamine-DNA adducts, especially those with an ortho substituent, may contribute to the biological activities of these compounds.

Full text of DOI:10.1021/tx950044z

Chem. Res. Toxicol. 1996, 9, 99-108
99
Syn th esis, Ch a r a cter iza tion , a n d Con for m a tion a l
An a lysis of DNA Ad d u cts fr om Meth yla ted An ilin es
P r esen t in Toba cco Sm ok e
M. Matilde Marques,*,† Lu´ısa L. G. Mourato,† M. Ame´lia Santos,† and
Frederick A. Beland*,‡
Centro de Quı´mica Estrutural, Complexo I, Instituto Superior Te´cnico, Av. Rovisco Pais,
1096 Lisboa Codex, Portugal, and Division of Biochemical Toxicology, National Center for
Toxicological Research, J efferson, Arkansas 72079
Received March 10, 1995X
The ability of a series of aromatic amines present in tobacco smoke (2-, 3-, and 4-methyl-
aniline, 2,3- and 2,4-dimethylaniline) to bind to DNA has been investigated by reacting
N-(acyloxy)arylamines with dG, dG nucleotides, and DNA. The predominant products from
reactions with dG and the nucleotides were characterized as N-(deoxyguanosin-8-yl)arylamines
by spectroscopic and HPLC methods. HPLC and spectroscopic analyses of the modified DNA
1
indicated the same adducts. Analyses of the H and 13C NMR spectra suggested that the
adducts containing a methyl substituent ortho to the arylamine nitrogen had a higher
percentage of syn conformers. This observation was supported by theoretical simulation studies
that indicated substantial percentages of low energy syn conformers, increasing with the
substitution pattern in the order para < meta < ortho < ortho,para < ortho,meta. The results
demonstrate that, although single-ring arylamines are considered weak carcinogens, their
electrophilic N-acetoxy derivatives, which are plausible metabolic intermediates, react with
DNA to yield covalent adducts structurally identical to those derived from carcinogenic
polyarylamines, such as 2-aminofluorene and 4-aminobiphenyl. Furthermore, the conforma-
tional perturbation induced in DNA by the formation of the monoarylamine-DNA adducts,
especially those with an ortho substituent, may contribute to the biological activities of these
compounds.
must undergo metabolic activation, via N-hydroxylation,
to reactive electrophilic intermediates that bind to DNA,
yielding covalent adducts (11, 12). In vitro and in vivo
studies have indicated that aromatic amine carcinogens
produce C8-deoxyguanosine-arylamine derivatives (dG-
C8-Ar)1 as predominant DNA adducts in nearly all
circumstances; N2-deoxyguanosine- and N6- and C8-
deoxyadenosine-arylamine adducts have also been re-
ported as minor products (11, 12). Arylamine-DNA
adducts are potential genotoxic lesions that have been
shown to induce mutations in bacterial and mammalian
test systems (refs 12 and 13 and references therein), as
well as in oncogenes of experimental animals (reviewed
in ref 12). Conformational perturbations associated with
a change from the normal anti to an abnormal syn
orientation about the glycosyl bond, accompanied by
stacking of the arylamine moiety with the nearby bases,
are thought to be decisive factors in the mutagenic and
tumorigenic properties of specific arylamine-DNA ad-
ducts (refs 14-19 and references therein).
In tr od u ction
Aromatic amines and amides are a class of chemical
carcinogens for which there is ample evidence of human
exposure (1). Although legal restrictions have curtailed
the magnitude of occupational hazard, the general popu-
lation remains exposed to aromatic amines from a variety
of ubiquitous sources. Cigarette smoke is one of the
major contemporary origins of exposure to these chemi-
cals, which are found in nanogram quantities in the
mainstream smoke and in higher concentrations in the
sidestream smoke (2, 3).
A wide range of epidemiological data indicates that
tobacco usage is causally related to human cancer in a
variety of organs, including the urinary bladder (4-6).
Compared to nonsmokers, cigarette users have been
estimated to be 2- to 10-fold more likely to contract
bladder cancer, depending on average daily usage and
cumulative consumption (7). Likewise, histologic changes
in the urinary bladder epithelium have been found to
correlate with smoking habits in a dose-responsive man-
ner (8). Among the profusion of chemical constituents
of tobacco smoke, only aromatic amines (e.g., 4-amino-
biphenyl and 2-naphthylamine) have been implicated as
human bladder carcinogens (9); therefore, arylamines are
regarded as determinant etiological factors in the induc-
tion of bladder cancer in smokers (10).
DNA adducts related to cigarette smoking have been
detected in various human tissues by sensitive tech-
niques, such as 32P-postlabeling (reviewed in ref 20).
Among these, several putative arylamine-DNA adducts
1
Abbreviations: Ar, arylamine; Bis-Tris, (bis[2-hydroxyethyl]imi-
notris[hydroxymethyl])methane; diMeA, dimethylaniline; dG, 2′-deoxy-
guanosine; dG3′p, 2′-deoxyguanosine 3′-monophosphate; dG5′p, 2′-
deoxyguanosine 5′-monophosphate; dG3′,5′p, 2′-deoxyguanosine 3′,5′-
bisphosphate; dG-C8-Ar, N-(deoxyguanosin-8-yl)arylamine; DEPT,
distortionless enhancement by polarization transfer; DMF, N,N-
dimethylformamide; dR, 2′-deoxyribosyl; dR5′p, 2′-deoxyribosyl 5′-
phosphate; FAB, fast atom bombardment; MeA, methylaniline; MD,
molecular dynamics; MM, molecular mechanics; NOE, nuclear Over-
hauser effect.
An extensive amount of evidence has demonstrated
that to generate their biological effects, aromatic amines
† Instituto Superior Te´cnico.
‡ National Center for Toxicological Research.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0893-228x/96/2709-0099$12.00/0 © 1996 American Chemical Society
+
+

100 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Marques et al.
of either 90 or 120 °C; the samples were dispersed in
thioglycerol matrix.
a
have been found in analyses of exfoliated cells and in
surgical autopsy and biopsy samples of human bladder
(21-24); however, with the exception of N-(deoxygua-
nosin-8-yl)-4-aminobiphenyl (22-24), none of the tobacco-
related adducts could be identified. Significantly higher
levels of 4-aminobiphenyl-hemoglobin adducts were also
found in blood samples from smokers, as compared to
nonsmokers (25).
1H NMR spectra were recorded on a Varian Unity 300 or a
Bruker AM 500 spectrometer, operating at 300 and 500 MHz,
respectively. 13C NMR spectra were recorded on a Varian Unity
300 spectrometer, operating at 75.43 MHz. The samples were
dissolved in Me2SO-d6 (2′-deoxyguanosine adducts) or Me2SO-
d6/D2O (2′-deoxyguanosine 5′-phosphate adducts). Proton chemi-
cal shifts were referenced by assigning the residual Me2SO
resonance to 2.49 ppm, and carbon chemical shifts, by assigning
the solvent resonance to 39.50 ppm. 1H resonance assignments
were based on comparison with literature data for 2′-deoxygua-
nosine (dG) and related dG adducts (36-38), combined with
homonuclear decoupling experiments, chemical exchange of the
labile protons with D2O, and observation of the nuclear Over-
hauser effect (NOE) enhancement patterns upon irradiation of
specific protons. 13C resonance assignments were based on
published data for dG (39) and related dG adducts (40), as well
as on the use of a distortionless enhancement by polarization
transfer (DEPT) pulse sequence and analysis of the 13C-1H
coupling patterns.
The major contribution to the arylamine content of
cigarette smoke comes from mononuclear arylamines,
such as aniline and its methyl and dimethyl derivatives
(2, 3). Although single-ring aromatic amines are gener-
ally regarded as weak carcinogens (26-28), the results
of a recent study of occupational carcinogenesis indicate
that chronic exposure to monoarylamines, in particular,
2-methylaniline (o-toluidine), may induce bladder cancer
in humans (29). Furthermore, hemoglobin adducts of
several single-ring arylamines present in tobacco smoke
have been detected in human blood samples, and in
certain cases (e.g., 2- and 4-methylaniline, 2,4-dimethyl-
aniline, and 2-ethylaniline), the adduct levels were
significantly higher in smokers than in nonsmokers (30,
31). The formation of these monoarylamine-hemoglobin
adducts implies the bioavailability of the corresponding
N-arylhydroxylamines (25, 30), thus suggesting that
single-ring arylamines undergo initial metabolic activa-
tion in a manner that parallels the activation of their
binuclear analogues. Therefore, mononuclear aromatic
amines may account for some of the putative arylamine-
DNA adducts detected in bladder samples from smokers
and may play a role in tobacco-induced carcinogenesis.
The preparation of well characterized monoarylamine-
DNA adduct standards would be of obvious importance
to test this hypothesis by allowing the unambiguous
identification and quantification of trace amounts of
potential adducts formed in vivo through comparison
with the synthetic standards (e.g., by 32P-postlabeling).
However, the difficulty in producing substantial quanti-
ties of DNA adducts from single-ring arylamines is
reflected in the limited number of successful syntheses
that have been reported (32-35).
Ch em ica ls. 2′-Deoxyguanosine was obtained from US Bio-
chemicals (Cleveland, OH). 2′-Deoxyguanosine 3′-monophos-
phate (dG3′p), 2′-deoxyguanosine 5′-monophosphate (dG5′p), 2′-
deoxyguanosine 3′,5′-bisphosphate (dG3′,5′p), salmon testes
DNA, and (bis[2-hydroxyethyl]iminotris[hydroxymethyl])methane
(Bis-Tris) were purchased from Sigma Chemical Co. (St. Louis,
MO). All other commercially available reagents were obtained
from Aldrich, through either Aldrich Chemical Co. (Milwaukee,
WI) or Sigma-Aldrich Qu´ımica, S. A. (Madrid, Spain), and were
used as received.
Syn th eses. Ca u tion : N-Arylhydroxylamines and their O-
acyl derivatives are potentially carcinogenic. They should be
handled with protective clothing in a well-ventilated fume hood.
Sta r tin g Ma ter ia ls. Pivaloyl cyanide [bp 119-120 °C, lit.
(41) 119-120 °C] was prepared according to the procedure of
Herrmann and Simchen (41).
N-Arylhydroxylamines [N-(2-methylphenyl)hydroxylamine,
mp 42-44 °C, lit. (42) 44 °C; N-(3-methylphenyl)hydroxylamine,
mp 67-69 °C, lit. (43) 68 °C; N-4-(methylphenyl)hydroxylamine,
mp 92-94 °C, lit. (43) 93-94 °C; N-(2,3-dimethylphenyl)-
hydroxylamine, mp 73-75 °C, lit. (42) 74 °C; and N-(2,4-
dimethylphenyl)hydroxylamine, mp 62-64 °C, lit. (44) 64.5 °C]
were synthesized in 50-60% yields by reduction of the corre-
sponding nitroarenes (10 mmol) at 50 °C with zinc dust (20-30
mmol) in aqueous ammonium chloride containing 30% ethanol
(42-45).
The N-arylhydroxylamines were converted to their O-acetyl
derivatives by an adaptation of the procedure of Lobo et al. (46,
47). Specifically, an argon-purged 200 mM solution of each
N-arylhydroxylamine in dry THF (2-5 mL), containing 1.1
molar equiv of triethylamine, was cooled to between -30 and
-40 °C. Acetyl cyanide (1.1 molar equiv) was then added, and
the mixture was stirred under argon for 0.5-1 h, while
maintaining the temperature below -30 °C. The unstable
N-acetoxyarylamines thus generated were kept in solution and
used immediately for the adduct syntheses. N-Pivaloxyaryl-
amines were prepared in a similar manner, using pivaloyl
cyanide as the acylating agent and a reaction time of 1.5-2 h
at -30 °C. As with the N-acetoxyarylamines, the N-piv-
aloxyarylamines were used immediately after synthesis for the
preparation of arylamine-dG adducts.
In the present study, we have investigated the ability
of several single-ring arylamines present in tobacco
smoke (2-, 3-, and 4-methylaniline, 2,3- and 2,4-dimethyl-
aniline) to form DNA adducts. We have characterized
the predominant products of reaction between electro-
philic synthons and dG, dG nucleotides, and DNA as dG-
C8-Ar adducts. In addition, we have conducted spectro-
scopic studies and energy minimization calculations to
establish the preferred conformations, which may deter-
mine the potential genotoxic effects of each adduct. The
results are discussed in relation to the number and
position of methylated substituents on the arylamine
ring.
Ma ter ia ls a n d Meth od s
N-(Deoxygu a n osin -8-yl)a r yla m in es. Meth od 1: By Re-
a ction of d G w ith N-Acetoxya r yla m in es. Typically, a cold
(-30 to -40 °C) THF solution containing the N-acetoxyaryl-
amine (generated from 1.1-1.2 molar equiv of the corresponding
N-arylhydroxylamine) was added to an argon-purged 120 mM
solution of dG in DMF/water (2/1), kept at 0-5 °C. The mixture
was further purged with argon and then stirred at room
temperature for 6-18 h. Following evaporation to dryness, the
residue was resuspended in water (10 mg of starting dG/mL)
and extracted 4-6 times with 1 volume of water-saturated
diethyl ether. The ether layer was discarded, and the aqueous
phase was extracted with 3 × 1 volume of water-saturated
1-butanol. The butanol extracts were combined and extracted
In str u m en ta tion . Melting temperatures were measured
with a Ko¨fler hot-stage apparatus and are uncorrected. Reversed-
phase HPLC analyses and separations were conducted using a
µBondapak C18 column (0.39 × 30 cm; Waters Associates,
Milford, MA) on a Waters Associates system consisting of two
Model 510 pumps, a U6K injector, and a Model 660 automated
gradient controller; the peaks were monitored at 280 nm with
a Hewlett-Packard 1040 diode array spectophotometric detector.
UV spectra were recorded with either a Beckman DU-40 or
a Shimadzu 1202 UV/vis spectrophotometer.
Fast atom bombardment (FAB) mass spectra were obtained
on a Finnigan TSQ 70 instrument, using a source temperature
+
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DNA Adducts from Methylated Anilines in Tobacco Smoke
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 101
with 1 volume of 1-butanol-saturated water to remove unreacted
dG. Following evaporation of the 1-butanol, the resulting
material was redissolved in 20% aqueous methanol. The dG-
C8-Ar adducts were subsequently isolated, in 1-1.5% average
yields, either by reversed-phase HPLC, using a 20-min linear
gradient of 20-70% aqueous methanol, followed by 5 min at
70% methanol, or by column chromatography on Sephadex LH-
20 (Pharmacia/PL Biochemicals, Piscataway, NJ ), using a 20-
80% step gradient of aqueous methanol. The spectral properties
of the products were fully consistent with the assigned struc-
tures (vide infra).
Met h od 2: By R ea ct ion of d G w it h N-P iva loxya r yl-
a m in es. Typically, a 1.5 M THF solution (3-6 mL) containing
the in situ generated N-pivaloxyarylamine was maintained at
-30 to -40 °C for 3-4 h. During this period, aliquots were
added at 30-min intervals to an argon-purged 200 mM solution
of dG in DMF/10 mM sodium citrate, pH 6 (2.5/1), kept at room
temperature. Upon addition of 4-8 molar equiv of the N-
pivaloxyarylamine, the mixture was allowed to react at room
temperature for an additional 16-18 h. After solvent extrac-
tions, the products were isolated by Sephadex LH-20 column
chromatography as described above. The following adducts,
structurally identical to those generated in method 1, were
obtained from the corresponding N-pivaloxyarylamines:
N-(Deoxygu an osin -8-yl)-2-m eth ylan ilin e (dG-C8-2-MeA).
η 3%; mp >220 °C dec; UV (EtOH) λmax 279 nm (log ꢀ 4.09); 1H
NMR (Me2SO-d6) δ 2.03 (1H, m, H2′′), 2.16 (3H, s, CH3), 2.69
(1H, m, H2′), 3.66 (2H, m, H5′,5′′), 3.87 (1H, m, H4′), 4.37 (1H,
m, H3′), 5.30 (1H, bs, 3′-OH), 5.49 (1H, bs, 5′-OH), 6.26 (1H, bs,
H1′), 6.29 (2H, bs, N2H2), 6.98 (1H, t, J 7.5 Hz, ArH4), 7.12 (1H,
t, J 7.5 Hz, ArH5), 7.17 (1H, d, J 7.2 Hz, ArH3), 7.31 (1H, d, J
7.2 Hz, ArH6), 7.92 (1H, bs, ArNH), 10.48 (1H, bs, N1H); 13C
NMR (Me2SO-d6) δ 17.86 (CH3), 38.02 (C2′), 61.23 (C5′), 71.02
(C3′), 82.88 (C1′), 87.19 (C4′), 112.35 (C5), 123.17 (ArCH), 126.02
(ArCH), 130.33 (ArCH), 139.13 (C8), 145.12 (ArC1/2), 147.79 (C4),
152.49 (C2), 155.56 (C6); m/ z (FAB) 373 (MH+, 40), 257 [(MH2
- dR)+, 84].
N-(Deoxygu an osin -8-yl)-3-m eth ylan ilin e (dG-C8-3-MeA).
η 3%; mp >250 °C dec; UV (EtOH) λmax 283 nm (log ꢀ 3.87); 1H
NMR (Me2SO-d6) δ 1.98 (1H, m, H2′′), 2.26 (3H, s, CH3), ∼2.50
(m, H2′, partially obscured by the solvent resonance), 3.73 (2H,
m, H5′,5′′), 3.90 (1H, m, H4′), 4.40 (1H, m, H3′), 5.32 (1H, bs,
3′-OH), 5.90 (1H, bs, 5′-OH), 6.30 (1H, bs, H1′), 6.34 (2H, bs,
N2H2), 6.43 (1H, d, J 7.5 Hz, ArH4), 7.12 (1H, t, J 7.8 Hz, ArH5),
7.48 (1H, s, ArH2), 7.54 (1H, d, J 7.5 Hz, ArH6), 8.53 (1H, bs,
ArNH), 10.53 (1H, bs, N1H); 13C NMR (Me2SO-d6) δ 21.37 (CH3),
38.34 (C2′), 61.25 (C5′), 71.22 (C3′), 82.74 (C1′), 87.12 (C4′),
112.16 (C5), 114.62 (ArCH), 117.78 (ArCH), 121.41 (ArCH),
128.39 (ArCH), 137.63 (C8), 140.76 (ArC1/3), 143.32 (ArC1/3),
149.48 (C4), 152.79 (C2), 155.68 (C6); m/ z (FAB) 395 [(M +
Na)+, 8], 373 (MH+, 26), 279 [(MH + Na - dR)+, 24], 257 [(MH2
- dR)+, 85].
(ArC1/2/3), 145.83 (ArC1/2/3), 149.90 (C4), 152.50 (C2), 155.63 (C6);
m/ z (FAB) 409 [(M + Na)+, 8], 387 (MH+, 100), 293 [(MH + Na
- dR)+, 6], 271 [(MH2 - dR)+, 96].
N-(Deoxygu a n osin -8-yl)-2,4-d im eth yla n ilin e (d G-C8-2,4-
d iMeA). η 9%; mp >220 °C dec; UV (EtOH) λmax 277 nm (log ꢀ
4.32); 1H NMR (Me2SO-d6) δ 2.03 (1H, m, H2′′), 2.12 (3H, s,
2-CH3), 2.23 (3H, s, 4-CH3), 2.69 (1H, m, H2′), 3.65 (2H, m,
H5′,5′′), 3.87 (1H, m, H4′), 4.37 (1H, m, H3′), 5.29 (1H, bs, 3′-
OH), 5.49 (1H, bs, 5′-OH), 6.26 (1H, bs, H1′), 6.26 (2H, bs, N2H2),
6.92 (1H, d, J 7.8 Hz, ArH5), 6.98 (1H, s, ArH3), 7.17 (1H, d, J
8.1 Hz, ArH6), 7.86 (1H, bs, ArNH), 10.45 (1H, bs, N1H); 13C
NMR (Me2SO-d6) δ 17.71 (CH3), 20.32 (CH3), 38.00 (C2′), 61.24
(C5′), 71.08 (C3′), 82.84 (C1′), 87.17 (C4′), 112.33 (C5), 123.83
(ArCH), 126.46 (ArCH), 130.84 (ArCH + ArC1/2/4), 132.45
(ArC1/2/4), 136.42 (C8), 145.59 (ArC1/2/4), 149.80 (C4), 152.42 (C2),
155.55 (C6); m/ z (FAB) 409 [(M + Na)+, 6], 387 (MH+, 40), 293
[(MH + Na - dR)+, 4], 271 [(MH2 - dR)+, 100].
N-(Deoxygu a n osin -8-yl)a r yla m in e n -P h osp h a tes (n ) 3′,
5′, or 3′,5′). Each nucleotide (dG3′p, dG5′p, or dG3′,5′p) was
dissolved in water to a concentration of 120 mM and reacted
overnight with 1.1-1.2 molar equiv of each specific N-
acetoxyarylamine, as described for the dG adducts. Following
evaporation of the THF, the aqueous solutions were further
diluted with water to a concentration of 30-40 mM and
extracted 4-6 times with 1 volume of water-saturated diethyl
ether to remove solvolysis products. For isolation of the dG5′p
adducts, the aqueous solutions were evaporated to dryness,
redissolved in 100 mM ammonium acetate (pH 5.7), and loaded
on Waters Sep-Pak C18 reversed-phase cartridges. The adducts
were subsequently separated by elution with a 0-50% step
gradient of acetonitrile in 100 mM ammonium acetate (pH 5.7).
The dG3′p adducts were isolated from the aqueous phase by
reversed-phase HPLC, using a 30-min linear gradient of 5-60%
acetonitrile in 100 mM ammonium acetate (pH 5.7). The
dG3′,5′p adducts were isolated by reversed-phase HPLC using
a similar gradient of 5-60% acetonitrile in 100 mM ammonium
acetate and 10 mM ammonium phosphate (pH 5.7). The
average adduct yields were estimated to be approximately 1%
on the basis of UV absorbance measurements and the molar
extinction coefficients determined for the corresponding dG
adducts. The dG5′p adducts, each prepared from 20 mg of the
starting nucleotide, were characterized by 1H NMR and mass
spectral analyses, as described below. In addition, these adducts
were treated with alkaline phosphatase for 3 h at 37 °C, and
the HPLC retention times and UV spectra of the resultant dG
adducts were compared to those of the dG adducts described
above. The dG3′p and dG3′,5′p adducts were prepared from 2.5
mg of the starting nucleotide. These adducts were identified
through comparison of their UV spectra with the spectra of the
dG and dG5′p adducts. The dG3′p and dG3′,5′p adducts were
also converted to dG adducts by similar incubations with
alkaline phosphatase, for HPLC and spectroscopic analyses.
N-(Deoxygu a n osin -8-yl)-2-m eth yla n ilin e 5′-P h osp h a te
(d G5′p -C8-2-MeA). 1H NMR (Me2SO-d6/D2O) δ 2.04 (1H, m,
H2′′), 2.15 (3H, s, CH3), 2.90 (1H, m, H2′), 3.75 (1H, m, H5′′),
3.85 (1H, m, H4′), 4.16 (1H, m, H5′), 4.54 (1H, m, H3′), 6.12
(1H, m, H1′), 6.87 (1H, t, J 7.3 Hz, ArH4), 7.08 (1H, t, J 7.7 Hz,
ArH5), 7.12 (1H, d, J 7.3 Hz, ArH3), 7.29 (1H, d, J 7.9 Hz,
ArH6); m/ z (FAB) 475 [(M + Na)+, 5], 453 (MH+, 14), 257 [(MH2
- dR5′p)+, 100].
N-(Deoxygu an osin -8-yl)-4-m eth ylan ilin e (dG-C8-4-MeA).
1
η 7%; mp >250 ° dec; UV (EtOH) λmax 284 nm (log ꢀ 4.42); H
NMR (Me2SO-d6) δ 1.98 (1H, m, H2′′), 2.22 (3H, s, CH3), ∼2.50
(m, H2′, partially obscured by the solvent resonance), 3.73 (2H,
m, H5′,5′′), 3.90 (1H, m, H4′), 4.39 (1H, m, H3′), 5.32 (1H, bs,
3′-OH), 5.89 (1H, bs, 5′-OH), 6.29 (1H, bs, H1′), 6.33 (2H, bs,
N2H2), 7.04 (2H, d, J 7.8 Hz, ArH3 + H5), 7.61 (2H, d, J 7.8 Hz,
ArH2 + H6), 8.53 (1H, bs, ArNH), 10.49 (1H, bs, N1H); 13C NMR
(Me2SO-d6) δ 20.35 (CH3), 38.40 (C2′), 61.35 (C5′), 71.36 (C3′),
82.85 (C1′), 87.21 (C4′), 112.14 (C5), 117.47 (ArC2,6/3,5), 128.92
(ArC2,6/3,5), 138.35 (C8), 143.62 (ArC1,4), 149.54 (C4), 152.82 (C2),
155.75 (C6); m/ z (FAB) 373 (MH+, 33), 257 [(MH2 - dR)+, 72].
N-(Deoxygu a n osin -8-yl)-2,3-d im eth yla n ilin e (d G-C8-2,3-
d iMeA). η 4%; mp >220 °C dec; UV (EtOH) λmax 280 nm (log ꢀ
4.07); 1H NMR (Me2SO-d6) δ 2.03 (4H, m + s, H2′′ + 2-CH3),
2.23 (3H, s, 3-CH3), 2.66 (1H, m, H2′), 3.65 (2H, m, H5′,5′′), 3.87
(1H, m, H4′), 4.37 (1H, m, H3′), 5.30 (1H, bs, 3′-OH), 5.50 (1H,
bs, 5′-OH), 6.27 (3H, bs, H1′ + N2H2), 6.92 (1H, d, J 7.2 Hz,
ArH4), 7.00 (1H, t, J 7.5 Hz, ArH5), 7.08 (1H, d, J 7.2 Hz, ArH6),
7.98 (1H, bs, ArNH), 10.50 (1H, bs, N1H); 13C NMR (Me2SO-d6)
δ 14.11 (CH3), 20.30 (CH3), 38.04 (C2′), 61.30 (C5′), 71.21 (C3′),
82.87 (C1′), 87.22 (C4′), 112.38 (C5), 122.17 (ArCH), 125.27
(ArCH), 125.42 (ArCH), 130.11 (ArC1/2/3), 136.85 (C8), 138.96
N-(Deoxygu a n osin -8-yl)-3-m eth yla n ilin e 5′-P h osp h a te
(d G5′p -C8-3-MeA). 1H NMR (Me2SO-d6/D2O) δ 2.04 (1H, m,
H2′′), 2.18 (3H, s, CH3), 2.78 (1H, m, H2′), 3.81 (1H, m, H5′′),
3.83 (1H, m, H4′), 4.16 (1H, m, H5′), 4.65 (1H, m, H3′), 6.17
(1H, m, H1′), 6.65 (1H, d, J 7.7 Hz, ArH4), 7.08 (1H, t, J 7.7
Hz, ArH5), 7.40 (1H, s, ArH2), 7.41 (1H, d, J 8.2 Hz, ArH6);
m/ z (FAB) 491 [(M + K)+, 10], 475 [(M + Na)+, 14], 453 (MH+,
16), 257 [(MH2 - dR5′p)+, 100].
N-(Deoxygu a n osin -8-yl)-4-m eth yla n ilin e 5′-P h osp h a te
(d G5′p -C8-4-MeA). 1H NMR (Me2SO-d6/D2O) δ 2.02 (1H, m,
H2′′), 2.21 (3H, s, CH3), 2.80 (1H, m, H2′), 3.81 (1H, m, H5′′),
3.86 (1H, m, H4′), 4.14 (1H, m, H5′), 4.62 (1H, m, H3′), 6.19
(1H, m, H1′), 7.01 (2H, d, J 8.4 Hz, ArH3 + H5), 7.54 (2H, d, J
8.4 Hz, ArH2 + ArH6); m/ z (FAB) 475 [(M + Na)+, 10], 453
(MH+, 24), 257 [(MH2 - dR5′p)+, 100].
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102 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Marques et al.
N-(Deoxygu a n osin -8-yl)-2,3-d im et h yla n ilin e 5′-P h os-
p h a te (d G5′p -C8-2,3-d iMeA). 1H NMR (Me2SO-d6/D2O) δ
2.01 (4H, s + m, H2′′+ 2-CH3), 2.22 (3H, s, 3-CH3), 2.93 (1H, m,
H2′), 3.73 (1H, m, H5′′), 3.83 (1H, m, H4′), 4.14 (1H, m, H5′),
4.56 (1H, m, H3′), 6.10 (1H, m, H1′), 6.82 (1H, d, J 7.5 Hz, ArH4),
6.96 (1H, t, J 7.5 Hz, ArH5), 7.02 (1H, d, J 7.5 Hz, ArH6); m/ z
(FAB) 489 [(M + Na)+, 3], 467 (MH+, 37), 271 [(MH2 - dR5′p)+,
100].
N-(Deoxygu a n osin -8-yl)-2,4-d im et h yla n ilin e 5′-P h os-
p h a te (d G5′p -C8-2,4-d iMeA). 1H NMR (Me2SO-d6/D2O) δ 2.03
(1H, m, H2′′), 2.10 (3H, s, 2-CH3), 2.20 (3H, s, 4-CH3), 2.90 (1H,
m, H2′), 3.74 (1H, m, H5′′), 3.85 (1H, m, H4′), 4.54 (1H, m, H3′),
6.10 (1H, m, H1′), 6.89 (1H, t, J 8.2 Hz, ArH5), 6.93 (1H, s,
ArH3), 7.18 (1H, d, J 8.2 Hz, ArH6); m/ z (FAB) 505 [(M + K)+,
7], 489 [(M + Na)+, 8], 467 (MH+, 10), 271 [(MH2 - dR5′p)+,
100].
F igu r e 1. Scheme for the synthesis of monoarylamine-DNA
adducts. R ) 2-Me, 3-Me, 4-Me, 2,3-diMe, or 2,4-diMe; R1 ) Me
or Me3C; R2 ) H or PO32-; Nu ) dG, dGnp (n ) 3′, 5′, or 3′,5′),
or DNA.
mental contaminants (1) and their potential role as
human carcinogens (9, 29), little is known about the
ability of mononuclear arylamines to undergo similar
transformations. Therefore, we selected a series of
single-ring arylamines present in tobacco smoke (2-, 3-,
and 4-methylaniline, 2,3- and 2,4-dimethylaniline) to
examine the capacity of suitably activated derivatives of
these amines to undergo DNA binding. Since the O-
acetylation of N-arylhydroxylamines has been implicated
as a major metabolic activation pathway for aromatic
amines (11), we tested N-acetoxyarylamines and their
synthetic equivalents, N-pivaloxyarylamines, as key
reactive intermediates. This strategy has been applied
previously by Boche and co-workers to the synthesis of
dG-C8-Ar adducts from the polyarylamine carcinogens
4-aminobiphenyl, 2-naphthylamine, and 2-aminofluorene
in low to moderate yields (50-52). The same investiga-
tors have shown that the procedure can be extended to
some monoarylamines, specifically aniline (33) and anilines
monosubstituted in the para position (34).
DNA Mod ifica tion Rea ction s. Chemical modification of
DNA with each of the arylamines was conducted using 500 µL
aliquots of a 1 mg/mL stock solution of salmon testes DNA in 5
mM Bis-Tris and 0.1 mM EDTA (pH 7.1). In some instances,
the DNA was dissolved in DMF/5 mM Bis-Tris and 0.1 mM
EDTA, pH 7.1 (65/35). Each aliquot was purged with argon and
reacted with 120 µL of a THF solution containing the N-
acetoxyarylamine, generated from 6-24 µmol of the correspond-
ing N-arylhydroxylamine, as described above. The mixture was
then incubated at 37 °C for 16-18 h. Following evaporation of
the THF, the aqueous solution was extracted sequentially with
3 × 1 volume of 1-butanol and 3 × 1 volume of diethyl ether,
both saturated with the same Bis-Tris buffer. The DNA was
precipitated by addition of 0.1 volume of 5 M NaCl and 1.1
volumes of absolute ethanol, washed with 70% ethanol, and
redissolved in 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1) to a
concentration of approximately 1 mg/mL. The arylamine-DNA
adducts in the treated samples were identified by comparison
of their HPLC retention times and UV spectra to those of the
corresponding dG-C8-Ar adducts, following standard enzymatic
hydrolysis of the DNA to nucleosides (48).
Th eor etica l Sim u la tion s. Energy minimization calcula-
tions were conducted with the molecular simulation program
Discover (version 2.9) from Biosym Technologies Inc., San Diego,
CA, using the AMBER forcefield, together with MOPAC charges
and distance-dependent dielectric constants to simulate the
solvent. The Biosym graphics program, Insight II (version
2.2.0), was used to design and display the structures. Molecular
dynamics (MD) was employed to generate different conforma-
tions, and molecular mechanics (MM) was used for refinement.
In each MD run, the temperature was 1000 K and the time step
was 1 fs. Two hundred structures were saved at intervals of
100 steps and then minimized using MM. No constraints were
imposed during the MD simulation to allow maximum freedom
of motion. Before each MD run, the starting structures for the
adduct were designed by using dG subunits in standard B-DNA
conformations and then minimized by MM and MOPAC.
Hydrogen bonds within the adduct molecules were also inves-
tigated. Since hydrogen positions are easily monitored in
theoretical studies, the hydrogen (H).. acceptor (A) distance was
monitored, instead of the donor (D)...acceptor (A) distance. The
normal criterion of 2.5 Å was taken as the upper limit for
effective hydrogen bonding, although less restrictive conditions
(e.g., 2.7 Å) have been used in some DNA simulations (49).
The calculated dihedral angles were defined as follows: R,
N9-C8-N(Ar)-C1(Ar); â, C8-N(Ar)-C1(Ar)-C2(Ar); ø, O4′-
C1′-N9-C4; γ, O5′-C5′-C4′-C3′ (Figure 1).
We prepared N-arylhydroxylamines by the classical
reduction of their nitro precursors with zinc dust (42-
45), and converted the reduced species to their N-acetoxy
and N-pivaloxy derivatives by reaction with acetyl cya-
nide and pivaloyl cyanide in dry THF at low temperature
(-30 °C to -40 °C). Although no attempts were made to
isolate the unstable N-(acyloxy)arylamines, which were
kept in solution and used immediately, the selective
O-acylation of N-arylhydroxylamines by acyl cyanides is
an established procedure in this temperature range (46,
47). As indicated in Figure 1, the N-(acyloxy)arylamines
were subsequently reacted with dG, dGnp (n ) 3′, 5′, or
3′,5′), and DNA. Following solvent extractions, to remove
side products of lower polarity, the adducts formed upon
reaction with dG (Figure 2a) and with the mononucleo-
tides were isolated by chromatographic techniques (vide
supra). The adducts formed upon reaction with DNA
were analyzed by HPLC (Figure 2b), after enzymatic
hydrolysis of the DNA to nucleosides. They were identi-
fied on the basis of their HPLC retention times and UV
spectra, by comparison with those of the adducts formed
upon reaction with dG. For all the monoarylamines
investigated, dG-C8-Ar adducts (vide infra) were identi-
fied systematically as the major products of reaction with
either the nucleoside, the nucleotides, or DNA. The
successful isolation of dG-C8-Ar adducts, using a syn-
thetic approach that simulates a plausible metabolic
pathway, suggests that methylated single-ring aryl-
amines have the potential to bind to DNA in vivo.
As a synthetic tool, the methodology followed in this
work is limited by the highly competitive solvolytic
decomposition of the N-(acyloxy)arylamines (53), which
restricts the adduct yields (33, 34, 50-52). We attempted
to overcome this obstacle by using a substantial molar
Resu lts a n d Discu ssion
Syn t h esis of Mon oa r yla m in e-DNA Ad d u ct s.
N-(Deoxyguanosin-8-yl)arylamine (dG-C8-Ar) adducts
are the major persistent species resulting from in vivo
covalent binding of electrophilic metabolites of carcino-
genic arylamines to DNA (11, 12) and are also the
predominant products formed in vitro by reaction of DNA,
dG, or dG nucleotides with model electrophilic synthons
(reviewed in ref 12). Despite their presence as environ-
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DNA Adducts from Methylated Anilines in Tobacco Smoke
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 103
F igu r e 3. NMR spectra of N-(deoxyguanosin-8-yl)-2,4-di-
methylaniline recorded at 22 °C. (a) 300 MHz 1H spectrum in
Me2SO-d6. The acquisition conditions were as follows: sweep
width, 6000 Hz; pulse width, 7 µs; acquisition time, 3 s;
relaxation delay, 0 s. “S” represents the residual Me2SO
resonance and “W” the water resonance. (b) 75.43 MHz 13C
spectrum in Me2SO-d6. The acquisition conditions were as
follows: sweep width, 40 000 Hz; pulse width, 6 µs; acquisition
time, 1 s; relaxation delay, 2 s. Low power broad-band decoup-
ling was accomplished by WALTZ-16 modulation. The time
domain data were treated with an exponential multiplication,
using a line broadening of 7 Hz. “S” represents the Me2SO
resonance.
F igu r e 2. HPLC of products obtained from (a) reacting
N-acetoxy-2,4-dimethylaniline with dG and (b) enzymatic hy-
drolysate of DNA after reaction with N-acetoxy-2,4-dimethyl-
aniline. The material eluting at 15 min was characterized as
N-(deoxyguanosin-8-yl)-2,4-dimethyaniline. The insets show the
UV spectra of the 15-min peaks.
and N-acetyl-N-(acyloxy)arylamines, which is quite suc-
cessful for the synthesis of dG-C8-Ar adducts from
carcinogenic polyarylamines (55-57), failed to produce
more than trace quantities of adducts from the monoaryl-
amines under investigation,2 possibly due to insufficient
stabilization of the presumed nitrenium/carbenium ion
intermediates by a single aromatic ring. On the other
hand, although an alternate method, based upon the
synthesis of N-(purin-8-yl)tolylamines from thiourea
derivatives (35), should have general applicability, the
problem of building the â-glycosyl bond to generate the
corresponding nucleosides/nucleotides is not straightfor-
ward. For example, the chemical coupling of adducted
bases to activated deoxyribose derivatives would require
multiple protecting steps (58), and previous attempts to
achieve enzymatic coupling have failed for guanine C8-
arylamine adducts (59).
excess of the N-(acyloxy)arylamines (up to ∼10 fold) but
experienced increased difficulties in the extraction of the
solvolysis products and the subsequent purification of the
adducts as the concentration of the N-(acyloxy)aryl-
amines was raised. Near-optimum conditions were
reached for dG modifications in DMF/water using a 4-8
molar excess of N-pivaloxyarylamines, presumably due
to lower rates of solvolysis in the presence of DMF and
to a slightly lower leaving ability of the bulky pivalate
group compared to acetate; even so, the adduct yields (3-
9%) were modest. Interestingly, the presence of a p-
methyl substituent in the arylamine ring (e.g., 4-methyl-
and 2,4-dimethylaniline) resulted in the more efficient
adduct syntheses, which suggests that the electron-
donating methyl substituent has a better ability to
stabilize the putative nitrenium/carbenium ion interme-
diate when located para to the electrophilic nitrogen. This
observation is consistent with previous theoretical work,
in which the enthalpies of nitrenium ion formation for a
series of alkylated anilines were estimated to be lower
for para-substituted anilines compared to their ortho- and
meta-substituted isomers (54).
Ch a r a cter iza tion of th e Ad d u cts. The major prod-
ucts of reaction between dG and the N-(acyloxy)aryl-
amines investigated were characterized as covalent ad-
1
ducts on the basis of UV, H and 13C NMR, and mass
spectral analyses (vide supra). Thus, the UV spectra
showed bathochromic shifts of 23-30 nm compared to
dG, as a result of increased conjugation, while the FAB
mass spectra were fully consistent with binding of each
arylamine fragment to the nucleoside. Likewise, the 13C
NMR spectra indicated the presence of all the dG and
the arylamine carbon resonances, as illustrated in Figure
3 (panel b) for the 2,4-dimethylaniline adduct (dG-C8-
2,4-diMeA).
Despite producing relatively low adduct yields, the
synthetic approach employed in this study emerges as
the only method currently available for the preparation
of monoarylamine-DNA adduct standards at the nucleo-
side/nucleotide level. The use of more stable electrophilic
synthons, such as N-acetoxy-N-(trifluoroacetyl)arylamines
2
L. L. G. Mourato and M. M. Marques, unpublished observations.
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104 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Marques et al.
Ta ble 1. Selected NMR Da ta for th e
N-(Deoxygu a n osin -8-yl)a r yla m in e (d G-C8-Ar ) a n d
N-(Deoxygu a n osin -8-yl)a r yla m in e 5′-P h osp h a te
(d G5′p -C8-Ar ) Ad d u ctsa
The structures of the dG adducts were established
1
upon analysis of their H NMR spectra, which indicated
that every isolated adduct was a dG-C8-Ar product. A
representative example is shown in Figure 3 (panel a)
for dG-C8-2,4-diMeA. In each case, all the dG proton
resonances were present, with the exception of the
nonexchangeable H8 at ∼δ 8.0; in addition, all the
arylamine ring proton resonances were observed, as well
as a 1-proton exchangeable resonance at δ 7.8-8.6
assigned to the arylamine NH. Moreover, the spectral
data for the 4-methylaniline adduct (dG-C8-4-MeA) were
in full agreement with those reported previously (38).
All the dG5′p adducts had FAB mass spectra in full
agreement with binding of arylamine fragments to the
adduct
δ (ppm)
Ar
H2′
C2′
dG
dG5′p
∼2.50b
40.33
NDc
∼2.56b
2-MeA
3-MeA
4-MeA
dG
dG5′p
2.69
2.90
38.02
NDc
dG
dG5′p
∼2.50b
38.34
NDc
2.78
dG
dG5′p
∼2.50b
38.40
NDc
2.80
1
nucleotide and H NMR spectra indicative of substitution
2,3-diMeA
2,4-diMeA
dG
dG5′p
2.66
2.93
38.04
NDc
of the amine nitrogen at the C8 of the guanine moiety.
In addition, upon treatment with alkaline phosphatase,
the dG5′p adducts were converted into products with
HPLC retention times and UV spectra identical to those
of the corresponding dG-C8-Ar adducts.
The quantities of isolated dG3′p and dG3′,5′p adducts
were insufficient for NMR analyses; nonetheless, the UV
spectra recorded for these products were identical to
those of the corresponding nucleoside and 5′-nucleotide
C8 adducts. Furthermore, the dG3′p and dG3′,5′p ad-
ducts were converted into corresponding nucleoside C8
adducts, as indicated by HPLC and UV spectral analyses,
upon incubation with alkaline phosphatase.
dG
dG5′p
2.69
2.90
38.00
NDc
a
The selected resonances correspond to the deoxyribose protons
and carbons that are sensitive to the glycosydic torsion angle. The
spectra were recorded at 22 °C in Me2SO-d6 (dG adducts) or
Me2SO-d6/D2O (dG5′p adducts). Proton chemical shifts were
referenced by assigning the residual Me2SO resonance to 2.49 ppm
and carbon chemical shifts by assigning the solvent resonance to
39.5 ppm. Partially obscured by the solvent resonance. c ND, not
b
determined.
the arylamine moiety induces a higher syn population.
The H NMR data for the dG5′p adducts (Table 1) are
1
The arylamine-DNA adducts were characterized after
enzymatic hydrolysis of the samples to nucleosides. In
each instance, the resultant products had HPLC reten-
tion times and UV spectra identical to the corresponding
dG-C8-Ar adducts (Figure 2).
fully consistent with this inference; thus, compared to
dG5′p, the H2′ resonances of the dG5′p adducts of
2-methylaniline, 2,3-dimethylaniline, and 2,4-dimethyl-
aniline were shifted downfield by ∼0.4 ppm, while the
analogous resonances for the adducts not containing an
o-methyl substituent were shifted in the same direction
to a lesser extent.
Con for m ation al P r oper ties of th e Addu cts. Among
the approaches applied for the estimation of the dynamic
anti a syn equilibria in nucleosides and nucleotides,
those based on inspection of the NMR chemical shifts of
the sugar H2′ and C2′ have been widely favored (36, 40,
60-62). Generally, a downfield shift of the H2′ resonance
and an upfield shift of the C2′ resonance are regarded
as indicators of an increased contribution by syn con-
former populations, with the H2′ chemical shifts being
more accurate for quantification (61). Quantitative
analyses have also been attempted on the basis of the
vicinal 13C-1H coupling constants between the sugar H1′
and the base carbons (40, 61, 63). Since the predominant
conformations about the glycosyl bond are thought to be
decisive factors for the mutagenic and carcinogenic
properties of specific dG-C8-Ar adducts, we conducted a
comparative analysis of the NMR spectral data for the
dG and the dG5′p adducts to search for any arylamine-
related conformational trends. Although the C8-H1′ and
C4-H1′ coupling constants were not sufficiently resolved
to allow systematic conclusions based on Karplus-type
relationships, some qualitative indications could be drawn
from the H2′ and C2′ chemical shifts, which are sum-
marized in Table 1.
Compared to dG, no changes were detected in the H2′
resonances of dG-C8-3-MeA and dG-C8-4-MeA. This
observation suggests that both adducts prefer anti con-
formations, in agreement with previous findings for the
4-methylaniline guanosine adduct (38). For all the
remaining dG adducts, the H2′ resonances exhibited
slight downfield shifts of similar magnitude (∼0.17 ppm)
relative to the analogous resonances in dG (Table 1).
Since these adducts have a methyl substituent ortho to
the amine function as a common structural feature, the
NMR data strongly indicate that ortho substitution in
13C NMR spectra were obtained on the dG adducts to
confirm the trends indicated by the 1H NMR spectra
(Table 1). Compared to dG, the C2′ resonance of dG-C8-
3-MeA and dG-C8-4-MeA was shifted upfield by ∼2 ppm,
while the analogous resonance in the other adducts, all
of which contained a methyl substituent ortho to the
amine function, was upfield by ∼2.3 ppm. The greater
upfield shift detected with the latter adducts is again
consistent with a greater contribution of syn conformers.
Th eor etica l Sim u la tion s. MD/MM techniques were
used to search for the minimum energy conformers of
each dG-C8-Ar adduct, with the purpose of probing the
conformational trends detected by NMR. Analysis of the
trajectory files (Figure 4) suggests that all the adducts
are quite flexible, as indicated in each case by a substan-
tial number of conformers having energies within 2.5
kcal/mol of the global minimum. The preferred geom-
etries of the glycosyl bonds, expressed as the frequencies
of occurrence of anti (ø ) 217-227°) and syn (ø ) 41-
45°) conformers found for the individual adducts in the
MD/MM simulations, are summarized in Table 2. Taking
into account conformers calculated along each MD/MM
run, the adducts had similar anti/ syn populations, with
anti conformers contributing in each case to approxi-
mately 60% of the total. However, differences became
apparent when the global minimum and the distributions
of the next lowest energy conformers (i.e., within 2 or 3
kcal/mol of the global minimum) were compared. Thus,
the 4-methylaniline adduct was found to adopt predomi-
nantly an anti conformation, having both an anti global
minimum and a high proportion (ca. 80%, within 3 kcal/
mol of the minimum) of low energy anti conformers. This
is consistent with previous calculations by Meier and
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DNA Adducts from Methylated Anilines in Tobacco Smoke
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 105
F igu r e 5. Diagrams of the lowest anti and syn energy
conformers calculated by MD/MM for the N-(deoxyguanosin-8-
yl)arylamine (dG-C8-Ar) adducts. Ar ) (a) 2-MeA; (b) 3-MeA;
(c) 4-MeA; (d) 2,3-diMeA; and (e) 2,4-diMeA. The dashed lines
represent the hydrogen bonds discussed in the text. See Table
3 for the energies and structural data.
F igu r e 4. Trajectory files of the total potential energy (kcal/
mol) versus time (ps) along the MD/MM runs for the N-(deoxy-
guanosin-8-yl)arylamine (dG-C8-Ar) adducts. Ar ) (a) 2-MeA;
(b) 3-MeA; (c) 4-MeA; (d) 2,3-diMeA; and (e) 2,4-diMeA. The
horizontal lines in each panel are drawn 2.5 kcal/mol above the
corresponding global minimum. Note that the energy scale
differs between panels.
The introduction of a second methyl substituent (e.g.,
2,3- and 2,4-dimethylaniline) resulted in global minima
having syn conformations and much higher percentages
of low energy syn contributors than those observed for
the monomethylated adducts. For instance, in the 2,4-
dimethylaniline adduct, only 58% of the conformers with
energies within 3 kcal/mol of the global minimum had
an anti geometry. This number decreased to 21% in the
2,3-dimethylaniline adduct, for which no anti conformers
were found within 2 kcal/mol of the global minimum.
Hence, the data in Table 2 suggest that the dG-C8-Ar
adducts derived from mono- and dimethylanilines will
exist as a weighted average of anti and syn conformers,
with the major contributors being clearly anti for the
4-methylaniline adduct and the percentage of syn con-
formers depending on the number of methyl substituents
and their position relative to the arylamine nitrogen
according to the sequence para < meta < ortho < ortho,
para < ortho,meta. These findings support the qualita-
Ta ble 2. F r equ en cies of Occu r r en ce of An ti a n d Syn
Con for m er s for th e N-(Deoxygu a n osin -8-yl)a r yla m in e
(d G-C8-Ar ) Ad d u cts a s Deter m in ed by MD/MM
Sim u la tion s
conformations
adduct
(dG-C8-Ar)
Emin+2b (%)
Emin+3c (%)
totald (%)
a
Ar
Emin
syn
anti
syn
anti
syn anti
2-MeA
3-MeA
4-MeA
2,3-diMeA
2,4-diMeA
syn
syn
anti
syn
syn
58
53
14
100
67
42
47
86
0
36
28
19
79
42
64
72
81
21
58
40
40
35
37
43
60
60
65
63
57
33
a
Conformation corresponding to the global minimum energy.
Conformations with energies within 2 kcal/mol of the global
b
minimum. c Conformations with energies within 3 kcal/mol of the
1
tive predictions drawn from analysis of the H and 13C
d
global minimum. Total conformations calculated along each MD/
NMR spectra. Interestingly, the results shown in Table
2 are not substantially different from those obtained by
computational methods for dG-C8-Ar adducts of bulkier
aromatic amines, such as the carcinogens 2-aminofluor-
ene and 4-aminobiphenyl, in which the theoretical pre-
diction of dynamic states containing a variety of low
energy anti and syn conformers (17, 64-69) has received
experimental confirmation from NMR studies of aryl-
amine-modified oligonucleotides (15, 16, 18, 19, 70-72).
MM run.
Boche (38), who determined an anti global minimum for
the analogous N-(guanosin-8-yl)-4-methylaniline adduct.
By contrast, both the 2- and 3-methylaniline adducts
were found to adopt syn global minima, although the
contribution of low energy anti conformers (64% and 72%,
respectively, within 3 kcal/mol of the global minimum)
was still predominant.
+
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106 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Marques et al.
Ta ble 3. En er gies a n d Str u ctu r a l Da ta Ca lcu la ted by MD/MM a n d Cor r esp on d in g to th e Globa l Min im a of both th e An ti
a n d Syn Con for m a tion s of th e N-(Deoxygu a n osin -8-yl)a r yla m in e Ad d u cts (d G-C8-Ar ) In vestiga ted
A...HBc (Å)
dihedral anglea (deg)
P1∧P2
b
adduct
Etotal
d1
d2
d3
(dG-C8-Ar) conformation (kcal/mol)
ø
γ
R
â
π
(O4′...HNAr) (N3...HO5′) (O5′...HNAr)
2-MeA
syn
anti
-19.21
-18.05
38.38
226.04 -176.67
57.25
-60.38 -51.03
-94.06 24.94
87.47
80.04
5.27
3.96
1.96
6.59
4.60
3-MeA
syn
anti
-25.23
-23.62
43.48
218.11
82.69
-61.76 -57.88
87.66
72.06
5.23
2.37
1.94
7.33
83.25 -119.12 -90.93
1.98
1.96
4-MeA
anti
syn
-25.51
-24.89
217.02
40.70
83.27 -117.33 74.59
75.97
88.10
2.36
5.25
7.32
3.15
79.52
-61.69 -56.90
2,3-diMeA
syn
anti
-22.44
-20.19
42.70
219.03
57.80
-55.84 -59.81
75.87
87.52
5.27
2.40
1.93
7.33
54.38 -118.32
57.20 -57.07 -51.68
-91.75 20.70
72.88
1.95
2,4-diMeA
syn
anti
-22.85
-21.37
44.78
227.45 -176.70
83.74
79.45
4.38
3.98
1.94
6.51
4.75
a
The dihedral angles are defined as follows: R, N9-C8-N(Ar)-C1(Ar); â, C8-N(Ar)-C1(Ar)-C2(Ar); ø, O4′-C1′-N9-C4; γ, O5′-
C5′-C4′-C3′ (Figure 1). Angle between the arylamine (P1) and the guanine (P2) planes. c Hydrogen (H)...acceptor (A) distances are
shown for the bonds indicated. Effective hydrogen bonding distances (i.e., <2.5 Å) are shown in bold.
b
The main structural parameters calculated for the
lowest energy anti and syn conformers of each adduct are
summarized in Table 3, and diagrams corresponding to
these conformers are displayed in Figure 5. The calcu-
lated data suggest that the occurrence of a stabilizing
effect due to hydrogen bonding between the sugar C4′-
oxygen and the arylamine NH will favor glycosydic
torsion angles in the anti domain, whenever the steric
hindrance is not too severe. This is the case with the
4-methylaniline adduct and the lowest anti conformer of
the 3-methylaniline adduct. However, ortho methylation
in the arylamine ring clearly introduces an unstabilizing
factor, which determines the preferred geometry of the
adduct and shifts the lowest energy conformers to the
syn domain. In addition, there is a clear relationship
between the order of increasing contribution of low
energy syn conformers (e.g., the monomethylated series)
and the order of increasing total energy. Interestingly,
even the geometry of the lowest energy anti conformers
is affected by ortho substitution. This is indicated by the
changes in the â dihedral angle observed for the 2-me-
thylaniline and 2,4-dimethylaniline adducts (Table 3),
which result in removal of the ortho methyl from the
vicinity of the sugar and prevent the establishment of
the hydrogen bond between O4′ and the arylamine NH
(Figure 5).
Con clu sion s
Aromatic amine carcinogens are typically activated
through N-hydroxylation, followed in some instances by
esterification, which creates a more reactive electrophile
(11, 12). In this study, we have demonstrated that
N-hydroxy derivatives of methylated anilines can be
converted synthetically into metabolically plausible N-
acetoxy products (and their N-pivaloxy equivalents) that
readily react with nucleosides (i.e., dG), nucleotides (i.e.,
dG3′p, dG5′p, and dG3′5′p), and DNA. In all cases, the
major adducts were characterized as C8-substituted dG
derivatives, which is consistent with what has been
observed with more carcinogenic aromatic amines, such
as 2-aminofluorene and 4-aminobiphenyl (11, 12). Al-
though C8-substituted dG adducts were the predominant
products in every instance, spectroscopic and theoretical
data indicated conformational differences depending
upon the location of the methyl substituent. Thus,
adducts containing a methyl group ortho to the amine
function (e.g., 2-methylaniline) had greater percentage
of syn conformers about the glycosyl bond than those not
bearing such a group. With other aromatic amines, the
occurrence of syn conformers has been associated with
both mutagenic and tumorigenic responses (14-19). In
this regard, it should be noted that aromatic amines
containing o-methyl substituents tend to be more mu-
tagenic (73, 74) and tumorigenic (75, 76) than analogues
with no substituent in the ortho position. This increase
in biological response may be due in part to the greater
propensity of ortho methylated adducts to adopt a syn
conformation.
An additional intramolecular hydrogen bond, involving
the C5′-oxygen and, again, the arylamine NH, was
detected in the lowest anti conformers of the 3-methyl-
aniline, 4-methylaniline, and 2,3-dimethylaniline adducts
(Table 3; Figure 5). This hydrogen bond, which has
also been found for the lowest energy conformer of
N-(guanosin-8-yl)-4-methylaniline (38) and for N-(deoxy-
guanosin-8-yl)-2-aminofluorene (36), appears to be re-
sponsible for the observed synclinal conformation of the
exocyclic C4′-C5′ bond that is normally preferred in
purine nucleotides. By contrast, in the lowest anti
conformers of the 2-methylaniline and 2,4-dimethyl-
aniline adducts, for which such hydrogen bonding is not
possible, the C4′-C5′ bond adopts an antiperiplanar
conformation. For all the syn conformers, with the
exception of the 4-methylaniline adduct, the calculations
indicated the establishment of an intramolecular hydro-
gen bond between the guanine N3 and the 5′-OH (Table
3). Such an interaction will obviously be absent at the
nucleotide level.
Ack n ow led gm en t. This work was supported in part
by NATO (Collaborative Research Grant CRG 910561)
and by J unta Nacional de Investigac¸a˜o Cient´ıfica e
Tecnolo´gica, Portugal (Contract PBIC/CEN/1154/92). We
thank J oanna Deck for some of the NMR spectra, J . Pat
Freeman for the FAB mass spectra, and Cindy Hartwick
for helping to prepare the manuscript. Part of this study
has appeared as a preliminary report (77).
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