Identification of New DNA Adducts of Phenylnitrenium

Article abstract of DOI:10.1021/acs.chemrestox.5b00120

Phenylnitrenium ion (PhNH⁺) may bind to nucleophiles through nitrogen as well as through C2 or C4 carbons. However, only adducts of the former type have been hitherto reported after its reaction with purine nucleosides. In this study, reactions of N-acetoxyaniline (PhNHOAc), a precursor to PhNH⁺, with 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG), and with DNA in vitro at physiological conditions are described. The reaction of PhNHOAc with dA followed by a hydrolytic deribosylation afforded 8-phenylaminoadenine (C8-PhNHA) together with a smaller amount of N⁶-(4-aminophenyl)adenine (N⁶-4APA). A similar reaction with dG afforded 8-phenylaminoguanine (C8-PhNHG) together with traces of 7-(4-aminophenyl)guanine (N7-4APG). The same adducts were found also in the DNA treated with PhNHOAc, and all of them were identified by comparison of their HPLC retention times and MS² spectra with a set of synthesized authentic adenine adducts at C2, C8, N7, and N⁶ positions and guanine adducts at C8, N7, and N² positions. The newly identified minor adduct, N7-4APG, represents the first proof of arylnitrenium adduction at the N7 position of dG, which is the prominent site of attack by most C-electrophiles.

Full text of DOI:10.1021/acs.chemrestox.5b00120

Article
pubs.acs.org/crt
Identification of New DNA Adducts of Phenylnitrenium
Antonín Kralík, Igor Linhart,* Lubomír Vana, and Alena Moulisova
́
́
́
̌
Department of Organic Chemistry, Faculty of Chemical Technology, University of Chemistry and Technology, Prague, Technicka
1905, CZ-166 28 Prague, Czech Republic
́
ABSTRACT: Phenylnitrenium ion (PhNH+) may bind to nucleophiles through
nitrogen as well as through C2 or C4 carbons. However, only adducts of the former
type have been hitherto reported after its reaction with purine nucleosides. In this
study, reactions of N-acetoxyaniline (PhNHOAc), a precursor to PhNH+, with 2′-
deoxyadenosine (dA), 2′-deoxyguanosine (dG), and with DNA in vitro at
physiological conditions are described. The reaction of PhNHOAc with dA followed
by a hydrolytic deribosylation afforded 8-phenylaminoadenine (C8-PhNHA) together
with a smaller amount of N6-(4-aminophenyl)adenine (N6-4APA). A similar reaction
with dG afforded 8-phenylaminoguanine (C8-PhNHG) together with traces of 7-(4-
aminophenyl)guanine (N7-4APG). The same adducts were found also in the DNA
treated with PhNHOAc, and all of them were identified by comparison of their HPLC retention times and MS2 spectra with a set
of synthesized authentic adenine adducts at C2, C8, N7, and N6 positions and guanine adducts at C8, N7, and N2 positions. The
newly identified minor adduct, N7-4APG, represents the first proof of arylnitrenium adduction at the N7 position of dG, which is
the prominent site of attack by most C-electrophiles.
namely, 7-(2-aminophenyl)-2′-deoxyguanosine (N7-2APdG)
and 7-(4-aminophenyl)-2′-deoxyguanosine (N7-4APdG), can
be expected. Despite numerous reports on arylnitrenium DNA
adducts derived from a wide variety of aromatic and
heteroaromatic amines as well as from nitroaromatics,1−5 to
our knowledge, no carbon-bound arylnitrenium adduct at the
guanine N7 position has been reported as yet. The lack of
information about this type of adduct may be due to the
unavailability of authentic samples of 7-(aminoaryl)guanines,
and these adducts may have escaped identification. To test our
hypothesis that arylnitrenium ions can form stable adducts also
by binding through one of the aromatic carbons at the N7
position of guanine and, possibly, also of adenine, we decided
to synthesize authentic samples of the expected phenyl-
nitrenium N7 adducts as well as some other possible adducts
with Ade and Gua. They were used to support our search for
adducts formed by the reaction of activated phenylhydroxyl-
amine as a source of PhNH+ with dG, dA, and DNA under
physiological conditions. These compounds (Chart 1) are
deribosylated analogues of dG and dA adducts and can
therefore be used as reference standards for the identification of
products obtained by the reaction of PhNHOAc with purine
nucleosides followed by hydrolytic treatment.
INTRODUCTION
■
Arylnitrenium ions are the ultimate mutagens, and carcinogens
formed by the dissociation of arylhydroxylamine esters with
sulfate and/or acetate in the course of the biotransformation of
many carcinogenic aromatic and heteroaromatic amines as well
as of nitroaromatics.1,2 Their main DNA binding site appears to
be the C8 position of guanine, but many minor adducts at other
sites such as C8 and N6 of adenine and N2 of guanine have also
been reported.3−5 In general, the C8 guanine position is a
favored site for radical attacks. In contrast, most electrophiles
react mainly at the N7 position of guanine.6,7 This certainly
holds for C-electrophiles, such as alkylating and arylating
agents. To explain the formation of the C8-N adducts, several
different mechanisms were proposed: (i) attack of the
nitrenium ion primarily at the N7 position with subsequent
migration of the arylamino group to C8 and deprotonation,8,9
(ii) direct attack of the nitrenium ion at C8 followed by
deprotonation,10 and (iii) primary attack by C2 of the
resonance stabilized nitrenium ion at N7 followed by an
imidazolidine ring closure and deprotonation accompanied by
ring reopening to yield the C8-N adduct as the end-product.11
The mechanism (iii), proposed by Parks et al., involves the
formation of the 7-(2-aminophenyl)guanine (N7-2APG)
intermediate, which in its protonated form rearranges to
8-phenylaminoguanine (C8-PhNHG). According to quantum
chemical calculations, C8-PhNHG is the most stable phenyl-
nitrenium adduct of guanine.11 The attack at N7 by an
electrophilic carbon atom seems to be very likely as the
arylnitrenium ions are resonance stabilized, and the charge is
delocalized through the aromatic system. In the case of
phenylnitrenium (PhNH+), which is the simplest model
arylnitrenium ion, nucleophiles may attack both the o- and
p-positions. Therefore, when reacting with dG two N7 adducts,
EXPERIMENTAL PROCEDURES
■
General. Polymer-bound 1,5,7-triazabicyclo[4.4.0]dec-5-ene
(TBD) was purchased from Novabiochem (Germany). 2′-Deoxy-
adenosine monohydrate and 2′-deoxyguanosine monohydrate (Fluka),
2-phenylaminoadenosine (Sigma), palladium catalyst (10 wt % Pd
on charcoal, Degussa type, Aldrich), and acetonitrile, LC/MS grade,
were purchased through Sigma-Aldrich, Prague, Czech Republic.
Received: March 20, 2015
Published: May 20, 2015
© 2015 American Chemical Society
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Triethylamine (TEA) was dried over calcium hydride, distilled, and
kept over molecular sieves. For LC/MS, distilled water was passed
through a Milli-Q water purification system (Millipore, Bedford, MA).
Phenylhydroxylamine (PhNHOH) was prepared according to Liu
et al.12 and N2-(dimethylaminomethylene)guanine (DMF-G) accord-
ing to Votruba et al.13 Other chemicals were of analytical or synthetic
grade and were used as received. LC/MS analyses were carried out on
a Thermo Scientific LXQ linear trap mass spectrometer in tandem
with a Janeiro LC system consisting of two Rheos 2200 pumps and a
CTC PAL autosampler. Electrospray ionization in the positive ion
mode was used. The collision gas was helium, the capillary tem-
perature was set to 300 °C, and the capillary voltage was 4.6 kV. The
collision energy was adjusted to achieve a nearly full fragmentation of
parent (M + H)+ ions using reference standards of the phenyl-
nitrenium adducts. NMR spectra were taken on a Varian Mercury
300 MHz spectrometer or a Bruker Avance 600 MHz spectrometer.
HRMS analyses were performed on an LTQ Orbitrap Velos
instrument (Thermo Fisher Scientific) using electrospray ionization
(ESI) in the positive mode.
Chart 1. Structures of Potential Adenine and Guanine
a
Adducts with PhNH+ Prepared as Reference Standards
Preparation of Authentic Purine Adducts. 7-(2-Aminophenyl)-
adenine (N7-2APA), 7-(4-aminophenyl)adenine (N7-4APA),14
8-phenylaminoadenine (C8-PhNHA),15 8-phenylaminoguanine (C8-
PhNHG),16 and 8-phenylamino-2′-deoxyguanosine (C8-PhNHdG)17
were prepared as reported in the literature.
7-(4-Aminophenyl)guanine (N7-4APG). DMF-G (288 mg,
1.4 mmol), 4-nitrophenylboronic acid (467 mg, 2.8 mmol), and
N,N,N′,N′-tetramethylethylenediamine (TMEDA) (420 μL, 325 mg,
2.8 mmol) were dissolved in dry methanol (20 mL) and Cu(II) acetate
(255 mg; 1.4 mmol), and activated molecular sieves 4 Å (1 g) were
then added to the solution. The resulting mixture was stirred at 45 °C
in an apparatus protected from moisture by a tube filled with
anhydrous calcium chloride. After 24 h, the reaction mixture was
diluted with chloroform (100 mL) and filtered through a thin layer of
Cellite. Column chromatography on silica gel afforded the crude
product containing a 5:1 mixture of isomeric 7 and 9-(4-nitrophenyl)-
guanine derivatives. Further chromatographic separation on silica gel
using a chloroform−methanol mixture as an eluent (the concentration
of methanol was increased gradually from 2 to 10% v/v) afforded
200 mg (44%) of 7-(4-nitrophenyl)-N2-(dimethylaminomethylene)-
guanine (N7-4NPDG) as a yellow powder. 1H NMR (DMSO-d6): δ =
3.04 and 3.18 (2 s, 3 + 3 H, NCH3); 7.94 (d, J = 9.1 Hz, 2H, Ar−H);
8.36 (d, J = 9.1 Hz, 2H, Ar−H); 8.53 (s, 1 H, 8-H); 8.69 (s, 1 H, N
CH); 11.63 (s, 1H, NH) ppm. 13C NMR (DMSO-d6): δ = 110.6
(C5); 125.0 and 125.8 (C2′, C3′, C5′, and C6′); 141.3 (C1′); 146.6
(C4′); 155.3 (C2); 158.1 (C6); 158.7 (C-amidine); 161.4 (C4) ppm.
HRMS (m/z): for C14H14N7O3 (M + H)+ calculated, 328.1153; found,
328.1154; for C14H13N7O3Na (M + Na)+ calculated, 350.0972; found,
350.0972.
Heating of N7-4NPDG (200 mg) in 30 mL of 1 M HCl for 5 h at
75 °C followed by neutralization with an aqueous ammonia solution
afforded a yellow precipitate, which was filtered off and washed with
a
The numbering of the purine ring is also shown.
Scheme 1. Synthesis of 7-Aminophenylguanines N7-2APG and N7-4APG
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Table 1. Diagnostic 13C NMR Signals of the N7-Arylguanine
Derivatives
chemical shift
compd
δ(C4) [ppm]
δ(C5) [ppm]
N7-2NPDG
N7-4NPDG
N7-2NPG
N7-4NPG
N7-2APG
N7-4APG
159.7
161.9
160.9
111.2
110.6
108.5
a
a
n.d.
n.d.
160.6
109.6
160.9
108.9
a
13C NMR spectrum could not be obtained due to the very low
solubility of N7-4NPG in DMSO.
Figure 1. LC-ESI-MS analyses of phenylnitrenium adducts arising
from the reaction of PhNHOAc with dA. Full scan ion chromatogram
of untreated reaction mixture (trace A) and extracted ion chromato-
grams at m/z 343 for MH+ of phenylnitrenium adducts with dA taken
before (trace B) and after hydrolysis by heating the reaction mixture
for 20 min at pH 7.4 to 100 °C (trace C).
Figure 2. LC-ESI-MS2 of phenylnitrenium adducts with adenine
obtained by the reaction of PhNHOAc with dA followed by acidic
hydrolysis (0.1 M HCl, 100 °C) taken at m/z 227 (top trace) as
compared with authentic C8-PhNHA, N6-4-APA, N6-2-APA, N7-
2APA, N7-4APA, and C2-PhNHA.
water. After drying over P4O10, 140 mg (86%) of 7-(4-nitrophenyl)-
guanine (N7-4NPG) as a yellow powder was obtained. 1H NMR
(DMSO-d6): δ = 6.33 (s, 2H, NH2); 7.91 (d, J = 9.1 Hz, 2H, Ar−H);
8.34 (d, J = 9.1 Hz, 2H, Ar−H); 8.44 (s, 1 H, 8-H); 10.99 (s, 1 H,
NH) ppm. HRMS (m/z): for C11H9N6O3 (M + H)+ calculated,
273.0731; found, 273.0733; for C11H8N6O3Na (M + Na)+ calculated,
295.0550; found, 295.0551.
13C NMR (DMSO-d6): δ = 108.6 (C5); 113.7 (C2′ and C6′); 125.0
(C4′); 125.9 (C3′ and C5′); 143.3 (C1′); 148.0 (C8); 153.2 (C2);
154.3 (C6); 160.9 (C4) ppm.
The nitro group of N7-4NPG was reduced by transfer hydro-
genation on palladium. The palladium catalyst (20 mg 10% Pd−C,
0.018 mmol) was activated by heating to 95 °C in a vacuum for
30 min. Dry methanol (12 mL), N7-4NPG (33 mg, 0.12 mmol), and
ammonium formate (500 mg, 8 mmol) were added, and the reaction
mixture was refluxed under argon. After 12 h, another portion of
ammonium formate was added (500 mg, 8 mmol), and the reaction
was continued for another 12 h. After cooling, the catalyst was filtered
off, washed thoroughly with hot water to remove excess ammonium
formate, and then extracted with DMSO. The extract was evaporated
to dryness in a vacuum, the residue triturated with water, filtered off,
and dried over P4O10. A yellowish powder (9 mg, 31%) was identified
as N7-4APG. 1H NMR (DMSO-d6): δ = 5.29 (s, 2H, NH2);
6.12 (s, 2H, NH2); 6.58 (d, J = 8.6 Hz, 2H, Ar−H); 7.13 (d, J =
8.2 Hz, 2H, Ar−H); 7.97 (s, 1H, 8-H); 10.71 (s, 1H, NH) ppm.
7-(2-Aminophenyl)guanine (N7-2APG). Synthesis of this
compound was accomplished in three steps as described for N7-
4APG. The ratio of 7- to 9-(2-nitrophenyl)guanine derivatives
obtained was 5:1. The yield of 7-(2-nitrophenyl)-N2-(dimethyl-
aminomethylene)guanine (N7-2NPDG) obtained as a yellow powder
1
was 165 mg (35%). H NMR (DMSO-d6): δ = 3.00 and 3.15 (2 s,
3 + 3H, NCH3); 7.79 and 7.90 (m, 1 + 1H, C4′-H and C5′-H); 8.24
and 8.27 (d, J = 9.1 Hz, 1 + 1H, C3′-H and C6′-H); 8.31 (s, 1 H,
8-H); 8.64 (s, 1 H, NCH); 11.47 (s, 1H, NH) ppm. 13C NMR
(DMSO-d6): δ = 111.2 (C5); 123.1 (C2′); 129.9, 130.1, and 130.4
(C3′, C4′, and C5′); 135.1 (C6′); 143.7 (C1′); 144.5 (C8); 155.3
(C2); 157.5 (C6); 158.4 (C-amidine); 159.7 (C4) ppm. HRMS
(m/z): for C14H14N7O3 (M + H)+ calculated, 328.1153; found,
328.1154; for C14H13N7O3Na (M + Na)+ calculated, 350.0972; found,
350.0974.
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Figure 3. LC-ESI-MS analyses of phenylnitrenium adducts arising
from the reaction of PhNHOAc with dG. Full scan ion chromatogram
(top trace) and extracted ion chromatogram at m/z 359 for MH+ of
phenylnitrenium adducts with dG (middle trace) as compared with
authentic 8-PhNHdG (bottom trace).
Acid hydrolysis of N7-2NPDG (1 M HCl, 5 h, 75 °C) afforded
7-(2-nitrophenyl)guanine (N7-2NPG) as an orange powder in 87%
1
yield. H NMR (DMSO-d6): δ = 6.63 (br s, 2H, NH2); 8.13 (d, J =
9.1 Hz, 2H, Ar−H); 8.24 (s, 1H, 8-H); 8.37 (d, J = 9.1 Hz, 2H,
Ar−H); 10.81 (br s, 1H, NH) ppm. 13C NMR (DMSO-d6): δ = 108.5
(C5); 126.1 (C2′); 129.9, 130.1, and 130.4 (C3′, C4′, and C5′); 135.2
(C6′); 143.4 (C1′); 144.4 (C8); 153.6 (C2); 154.5 (C6); 157.0 (C-
amidine); 160.9 (C4) ppm. HRMS (m/z): for C11H9N6O3 (M + H)+
calculated, 273.0731; found, 273.0732; for C11H8N6O3Na (M + Na)+
calculated, 295.0552; found, 295.0556.
Transfer hydrogenation of N7-2NPG under the same conditions as
those described for N7-4NPG afforded 6 mg (21%) of yellowish
powder, which was identified as N7-2APG. 1H NMR (DMSO-d6): δ =
4.94 (s, 2H, NH2); 6.12 (s, 2H, NH2); 6.55−6.59 (m, 1H, Ar−H);
6.78 (d, J = 7.4 Hz, 1H, Ar−H); 6.98−7.01 (m, 1H, Ar−H); 7.09−
7.13 (m, 1H, Ar−H); 7.85 (s, 1H, 8-H); 10.76 (s, 1H, NH) ppm. 13C
NMR (DMSO-d6): δ = 109.6 (C5); 116.1 and 116.2 (C3′, C6′); 121.9
(C2′); 128.5 and 129.8 (C4′ and C5′); 144.1 (C6′); 143.4 (C1′);
145.1 (C8); 153.4 (C2); 154.3 (C6); 160.6 (C4) ppm.
N6-(4-Aminophenyl)adenine (N6-4APA). 6-Chloropurine
(310 mg, 2 mmol), p-phenylenediamine (430 mg, 4 mmol), and
TEA (556 μL, 404 mg, 4 mmol) were refluxed in 15 mL of anhydrous
1-propanol for 3 h. The starting material was dissolved upon heating,
and 30 min later, the product began to precipitate. After 3 h, the
reaction mixture was allowed to cool, and the greyish precipitate was
filtered off, washed with ethanol, and dried over P4O10. The crude
product (434 mg, 96%) was recrystallized from aqueous ethanol to
give 290 mg of (63%) of N6-4APA. 1H NMR (DMSO-d6): δ = 6.55 (d,
J = 8.5 Hz, 2H, Ar−H); 7.42 (d, J = 8.5 Hz, 2H, Ar−H); 8.15 (s, 1H,
C2−H); 8.21 (s, 1H, C8−H); 4.90 (bs, 2H, NH2); 9.25 (bs, 1H, NH);
13.0 (bs, 1H, NH) ppm. 13C NMR (DMSO-d6): δ = 114.2 (C2′ and
C6′); 119.5 (C5); 123.3 (C3′ and C5′); 129.1 (C4′); 139.9 (C4);
145.0 (C1′); 150.4 (C2); 152.5 (C8) ppm.
Figure 4. LC-ESI-MS2 of phenylnitrenium adducts with guanine
obtained by the reaction of PhNHOAc with dG followed by neutral
hydrolysis (100 °C, 20 min). Extracted ion chromatogram taken at
m/z 243 (top trace) as compared with authentic C8-PhNHG,
C8-PhNHdG, N7-4APG, and N7-2APG, and N2-4APG and N2-2APG.
powder, which was identified as N6-2APA. 1H NMR (DMSO-d6): δ =
6.60 (t, J = 7.7 Hz, 1H, Ar−H); 6.76 (d, J = 8.2 Hz, 1H, Ar−H); 6.93
(t, J = 7.6 Hz, 1H, Ar−H); 7.26 (d, J = 7.9 Hz, 1H, Ar−H); 8.17 (C2−
H); 8.18 (C8−H); 5.82 (bs, 2H, NH2); 8.90 (bs, 1H, NH) ppm. 13C
NMR (DMSO-d6): δ = 116.7 and 117.1 (C3′ and C5′); 120.0 (C5);
125.2 (C2′); 126.5 and 127.4 (C4′ and C6′); 143.9 (C1′); 150.5
(C2); 152.8 (C8); 153.9 (C6) ppm.
N6-(2-Aminophenyl)adenine (N6-2APA). The reaction of
6-chloropurine with o-phenylenediamine under the conditions
described above for N6-4APA proceeded more slowly, so that it was
complete after 8 h. No precipitate was spontaneously formed.
Therefore, the solvent was evaporated in a vacuum, and the residue
triturated with water. The product (230 mg, 51%), which remained
undissolved, was filtered off, washed with ethanol, and dried over
P4O10. Crystallization from ethanol afforded 85 mg (19%) of yellowish
N2-(4-Aminophenyl)guanine (N2-4APG). 2-Bromohypoxanthine
(215 mg, 1 mmol), p-phenylenediamine (324 mg, 3 mmol), and TEA
(73 μL, 101 mg, 1 mmol) were dissolved in 10 mL of dry 1-propanol,
and the reaction mixture was refluxed for 4 h. After cooling, the
precipitate formed was filtered off and washed thoroughly with
aqueous ethanol followed by pure ethanol. After drying over P4O10,
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Scheme 2. Formation of Phenylnitrenium Adducts by the
Reaction of PhNHOAc with dG and dA
Figure 5. LC-ESI-MS2 analyses of phenylnitrenium adducts with
adenine in the DNA reacted with PhNHOAc. Extracted ion
chromatogram of acid hydrolyzed DNA (top trace) taken at m/z
210 from MS2 of the parent ion m/z 227 (MH+) as compared with
with authentic N6-4APA and C8-PhNHA.
C8−H); 7.64 (bs, 1H, NH); 10.45 (bs, 1H) ppm. 13C NMR (DMSO-
d6): δ = 108.4 (C5); 111.6; 116.3 and 116.8 (C3′-6′); 126.3 (C1′);
151.7 and 153.3 (C4 and C6) ppm.
2-Phenylaminoadenine (C2-PhNHA). 2-Phenylaminoadenosine
(10 mg, 0.028 mmol) was dissolved in 0.1 M HCl (4 mL) and stirred
at 80 °C for 4 h. The solution was then allowed to stand overnight at
room temperature. The white precipitate, which was formed upon
neutralization with NaOH to pH 7, was filtered off, washed with water,
and dried over P4O10. The product was obtained as a white powder
(4 mg, 63%). 1H NMR (DMSO-d6): δ = 6.82 (t, J = 7.2 Hz, 2H, Ar−
H); 7.20 (t, J = 7.6 Hz, 2H, Ar−H); 7.82 (d, J = 7.8 Hz, 1H, Ar−H);
8.74 (s, 1H, C8−H); 6.79 (s, 1H, NH); 7.79 (s, 1H, NH); 12.4 (s, 1H,
NH) ppm. 13C NMR (DMSO-d6): δ = 114.0 (C5); 118.0 (C2′ and
C6′); 119.8 (C4′); 128.1 (C3′ and C5′); 136.4 (C8); 141.8 (Ar−C);
151.4 (C4); 155.7 (C2); 156.7 (C6) ppm.
Reaction of N-Acetoxyaniline (PhNHOAc) with Purine
Nucleosides. PhNHOH (4 mg, 0.04 mmol) was dissolved in 2 mL
of dry tetrahydrofuran (THF), polymer-bound TBD (40 mg,
0.116 mmol) and pyruvonitrile (20 μL, 19 mg, 0.28 mmol) were
added, and the resulting mixture was stirred for 10 min at room
temperature. An aliquot of this solution (0.8 mL) containing activated
PhNHOAc was added to a solution of corresponding nucleoside, i.e.,
dA (5.2 mg, 0.02 mmol) or dG (6 mg, 0.02 mmol) in 0.1 M phosphate
buffer, pH 7.4 (6 mL). The reaction mixture was incubated at 37 °C
for 1 h. In the course of incubation, the other two PhNHOAc aliquot
solutions of 0.6 mL were subsequently added after 15 and 30 min.
Samples of the reaction mixture were then hydrolyzed under neutral
and acidic conditions. For the neutral hydrolysis, an aliquot of 2 mL
was refluxed for 20 min. For the acidic hydrolysis, aliquots were mixed
with equal volumes of 0.1 M HCl and heated at 60 or 100 °C for
20 min.
Figure 6. LC-ESI-MS2 analyses of phenylnitrenium adducts with
guanine in the DNA reacted with PhNHOAc. Extracted ion
chromatogram of acid hydrolyzed DNA (top trace) taken at m/z
226 from MS2 of the parent ion m/z 243 (MH+) as compared with
authentic N7-4APG and C8-PhNHG.
the product was obtained as a slightly greyish powder (160 mg, 66%).
1H NMR (DMSO-d6): δ = 4.95 (bs, 2H, NH2); 6.52 (d, J = 8.3 Hz,
2H, C3′-H and C5′-H); 7.15 (d, J = 8.0 Hz, 2H, C2′-H and C6′-H);
8.1 (bs, 1H, NH); 10.3 (bs, 1H, NH) ppm. 13C NMR (DMSO-d6-
DCl-D2O): δ = 109.5 (C5); 121.6 and 124.4 (Ar−CH); 126.5 (C1′);
137.1 (C2); 138.3 (C4′); 149.3, 151.7, and 153.3 (C4, C6 and C8)
ppm.
N2-(2-Aminophenyl)guanine (N2-2APG). N2-2APG was pre-
pared as described for N2-4APG using o-phenylenediamine instead of
its p-isomer and 1-butanol instead of 1-propanol. The crude product
obtained (70 mg, 33%) was recrystallized from ethanol to afford 20 mg
Reaction of N-Acetoxyaniline (PhNHOAc) with DNA. Calf
thymus DNA (2.5 mg) was dissolved in 3 mL of a buffer solution
consisting of 10 mM TRIS/HCl and 1 mM EDTA (pH 7.4).
PhNHOAc solution in THF (1.5 mL), which was prepared from 3 mg
(0.03 mmol) of PhNHOH, 15 mg (0.22 mmol) of pyruvonitrile,
and 30 mg of polymer-bound TBD as described above, was added
1
(9%) of a beige powder. H NMR (DMSO-d6): δ = 4.95 (bs, 2H,
NH2); 6.55 (t, J = 7.9 Hz, 1H, C5′-H); 6.72 (d, J = 7.1 Hz, C6′-H);
6.90 (t, J = 8.1 Hz, C3′-H); 7.38 (d, J = 7.1 Hz, C3′-H); 7.62 (s, 1H,
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a
Table 2. Mass Spectra (ESI) of Potential Adenine and Guanine Adducts of Phenylnitrenium
(M+H)+ (m/z)
formula
fragments (m/z)
found Δ mmu
210
abundance
[%]
formula
compd
found
Δ mmu
−0.1
C8-PhNHA
227.1039
227.1040
227.1040
C11H11N6
100
30
5
C11H8N5
C10H10N5
C10H9N4
C11H8N5
C10H10N5
C10H9N4
C11H8N5
C10H10N5
C10H9N4
C11H8N5
C10H10N5
C11H8N5
C10H10N5
C9H8N5
200
185
N7-2APA
N7-4APA
0.0
C11H11N6
C11H11N6
210
210
100
5
200
200
185
185
7
+0.0
210.0774
200.0931
185.0822
210.0774
200.0931
210.0781
200.0937
186.0781
134.0717
108.0688
210
−0.1
0.0
100
10
5
−0,2
+0.7
+0.6
+0.7
+0.6
+0.7
+0.5
+0.6
N6-2APA
N6-4APA
227.1041
227.1042
+0.1
+0.2
C11H11N6
C11H11N6
100
28
100
30
7
22
7
C7H8N3
C6H8N2
C2-PhNHA
C8-PhNHG
N7-2APG
227.1044
243.0991
243.0986
+0.4
+0.2
−0.3
C11H11N6
100
3
C11H8N5
C10H10N5
C11H8N5O
C10H9N4O
C11H8N5O
C10H9N4O
C10H10N5
C9H8N3
200
C11H11N6O
C11H11N6O
226
100
18
100
42
15
8
201
226.0723
201.0771
200.0934
158.0712
226.0723
201.0770
226.0716
134.0708
226.0717
−0.1
0.0
+0.3
−0.1
0.0
N7-4APG
N2-2APG
N2-4APG
243.0989
243.0985
243.0983
0.0
C11H11N6O
C11H11N6O
C11H11N6O
100
33
100
10
100
C11H8N5O
C10H9N4O
C11H8N5O
C7H8N3
0.0
−0.5
−0.7
−0.7
−0.5
−0.6
C11H8N5O
a
Quasimolecular ions (MH+) and product ions in the collision induced MS2 taken at m/z of respective MH+ parent ions.
the nitro group (Scheme 1). The protecting group at N2 was
required to redirect the arylation reaction from N9 to N7
position. This reaction has been previously described for
methyl- and vinylphenylboronic acids to give mainly N7-
arylguanines accompanied by minor amounts of N9-isomer
(N7/N9 isomer ratios up to 6:1).18 We have newly observed a
similar selectivity (5:1) for arylation with nitrophenylboronic
acids, although this reaction required a somewhat elevated
temperature. To distinguish between N7- and N9-substituted
products, diagnostic signals of C4 and C5 in 13C NMR spectra
were used (Table 1). For N7-arylguanines, C4 and C5 res-
onances should be around 160 and 110 ppm, respectively, as a
result of an anisotropic effect caused by aryl substitution.14,18
Reaction of dA with N-Acetoxyaniline (PhNHOAc).
PhNHOH can be activated for reactions with nucleophiles by
acetate ester formation. Arylhydroxylamine esters are also
formed in the course of the metabolism of many mutagenic and
carcinogenic arylamines as well as nitroaromatics. Their
reactions with nucleosides are relevant simplified models of
DNA adduct formation in vivo. Although, to our knowledge, the
reaction of PhNHOAc or another active ester of PhNHOH
with dA itself has not been reported in the literature, it is
known that similar reactions, such as that of PhNHOH
benzoate ester with adenosine19 or other arylhydroxylamine
esters with dA, give mainly corresponding C8- and/or
N6-adenine adducts.20,21 We have detected two major adducts
at m/z 343 arising from the reaction of PhNHOAc with dA at
pH 7.4 and 37 °C (Figure 1). Conversion of dA to these
subsequently in three equal portions, and the reaction mixture was
incubated for 1 h under gentle stirring. The time interval between
individual additions was 15 min. To release the adducts formed, the
solution was refluxed for 40 min and analyzed by LC/MS.
Analyses of the Adducts with PhNH+ by LC/MS. Samples
hydrolyzed under acidic conditions were neutralized to pH 7. Both
hydrolyzed and nonhydrolyzed samples of the reaction mixtures were
diluted 10 times with 10% aqueous acetonitrile, and aliquots of 10 μL
were injected onto a 150 × 2 mm Phenomenex Aqua C18 column,
5 μm particle size, which was eluted with 0.1% formic acid in aqueous
acetonitrile at the flow rate of 0.2 mL/min. For dA and Ade adducts,
the concentration of acetonitrile was increased linearly from 0 to 60%
within 20 min. For dG and Gua adducts, a two-step gradient of
acetonitrile was used, i.e., from 0 to 30% within 15 min and thereafter
to 50% in another 5 min. The mass analyzer was set to monitor MS2
spectra at m/z values corresponding to (M + H)+ of the parent ions
(m/z 343.1, 359.1, 243.1, and 227.1 for dA, dG, Gua, and Ade adducts,
respectively). In addition, full scan spectra were also taken in the range
of m/z 100−400. Conversion of dA to the major adducts found was
estimated from the peak areas taken from extracted mass chromato-
grams at respective m/z values of the (M + H)+ ions.
RESULTS AND DISCUSSION
■
Synthesis of the N7-Guanine Adducts. To prove
whether N7-guanine adducts with phenylnitrenium ion are
formed upon the reaction of dG with PhNHOAc, authentic
samples of the expected adducts were needed. These samples
were obtained by arylation of protected guanine (DMF-G) with
2- and 4-nitrophenylboronic acids followed by hydrolytic
removal of the amidino protecting group and reduction of
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adducts was approximately 0.6%, and their ratio was about 2:1.
After acidic hydrolysis, two major deribosylated adducts were
obtained, which were identified as C8-PhNHA and N6-4APA
by LC/MS analysis by comparing their retention time and
MS2 spectra with those of synthesized reference standards
(Figure 2). Other synthesized authentic adducts, i.e., C2-
PhNHA, N7-2APA, N7-4APA, and N6-2APA, were also
searched for, but none of them coeluted with any of the
minor peaks detected at m/z 227 (Figure 2). The formation of
N6-4APA can be explained by the SN1 mechanism proceeding
via the phenylnitrenium ion rather than by SN2 reaction of
PhNHOAc with dA. Although it is known that PhNH+ has a
very short lifetime of 0.25 ns in aqueous solutions,22,23 it is
most likely the ultimate electrophile attacking the nucleophilic
amino group at C6. Surprisingly, despite its short lifetime, it is
able to react rather selectively with the most nucleophilic
reaction center of dA (Scheme 2).
Reaction of dG with PhNHOAc. In accordance with
previous reports,1,24 the reaction of PhNHOAc with dG under
physiological conditions afforded C8-PhNHdG as the predom-
inating adduct. Its identity was confirmed by a comparison of
its elution time and MS2 spectra with an authentic sample
(Figure 3). The conversion of dG to 8-PhNHdG amounted to
about 3.5% as estimated by comparing corresponding peak
areas in extracted ion chromatograms. After hydrolysis by
heating at 60 or 100 °C for 20 min, several peaks at m/z 243
corresponding to MH+ of deribosylated PhNH+ adducts were
detected. Among them, C8-PhNHG and N7-4APG were
identified using authentic standards (Figure 4). Another
expected N7-guanine adduct, N7-2APG, has not been detected.
Deoxyguanosine adducts at the N7 position are prone to
hydrolytic deribosylation due to the weakening of the glycosidic
bond caused by the positively charged imidazolium ring.
Therefore, unlike C8-arylamino adducts, the N7-adducts can be
effectively deribosylated even under the conditions of mild
neutral hydrolysis.7,25 It is therefore not surprising that after
neutral hydrolysis, only a portion of C8-PhNHdG was
deribosylated to C8-PhNHG (Figure 4). Complete deribosy-
lation of dG adducts can be usually achieved by acidic
hydrolysis. When heating the reaction mixture in 0.05 M HCl at
60 or 100 °C, the adducts with dG underwent a degradation, so
that at least 10 peaks at m/z 243 (MH+ of PhNH+ adducts with
Gua) were found in the extracted ion mass chromatograms.
These results show that PhNHOAc can react with dG not only
at C8 position but also at other nucleophilic sites including the
N7 position. However, only a small amount of the N7-adduct is
formed as compared with the C8-adduct, which was the only
major adduct formed (Scheme 2). Adducts at the exocyclic
NH2 group, namely, N2-2APG and N2-4APG, have not been
detected (Figure 3). This type of adduct is known to be formed
by some other arylnitrenium ions.1
found in the reactions with dA and dG, namely, C8-PhNHG,
C8-PhNHA, and N6-4APA. It cannot be excluded that at least a
part of these adducts might have originated from binding to
nucleoside and/or nucleotide fragments formed in the course
of incubation rather than from untouched DNA.
Mass Spectra of Adducts. The deribosylated adducts
formed by the above-mentioned reaction of PhNHOAc
were identified by a comparison with synthesized authentic
standards. Retention times and MS2 spectra of the reference
standards were used to support identification. Despite certain
common features in MS fragmentation, isomeric adducts also
showed some distinctive fragments, which facilitated structural
assignment. Compositions of the key fragment ions were
determined unequivocally by HRMS. Results are listed in
Table 2, and proposed fragment structures for N6-4APA are
shown in Scheme 3 and those of N7-2APG and N7-4APG in
Scheme 3. Fragmentation of N6-4APA in Collision Induced
MS2 Taken at m/z 227 for MH+
Reaction of PhNHOAc with DNA. The same PhNH+
adducts as those identified in the reactions of PhNHOAc with
dA and dG, albeit at low concentrations, were detected after its
reaction with the DNA in vitro under physiological conditions
followed by neutral hydrolysis. These adducts were also
identified by comparison of their retention times and MS2
spectra with those of reference standards. Mass chromatograms
are shown in Figures 5 and 6. Neutral hydrolysis was chosen
because the N7-guanine adducts are known to be easily cleaved
from the DNA due to destabilization of the N-glycosidic
bond.7,25 However, after mild neutral hydrolysis we could
detect not only N7-4APG but also all other adducts previously
Scheme 4. A strong tendency toward cyclization leading to
heteroaromatic structures seems to be a characteristic feature of
all tested adducts.
Mechanistic Considerations. A detailed mechanism of the
reaction of arylhydroxylamine esters with purine nucleosides
has been a subject to dispute over the years. Several proposals
were based on the primary attack of the positively charged
nitrogen of the nitrenium ion at the nucleophilic N7 position of
dG or other nucleosides.1 More recently, McClelland et al.
proposed that this reaction proceeds as a straightforward
electrophilic substitution, i.e., substitution of ArNH+ for
H+ at the C8 position of dG. This mechanism was strongly
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Scheme 4. Fragmentation of N7-APG in Collision Induced MS2 Taken at m/z 243 for MH+
Notes
supported by identification of the key intermediate, a primary
arylnitrenium adduct at the C8 position of dG.10 These adducts
were found to be the predominating DNA adducts for all
arylnitrenium ions tested, but other minor ones, namely, those
at C8 and N6 of adenine and N2 of guanine, have also been
reported.1 Binding of arylnitrenium ions through both nitrogen
and carbon atoms can be explained by a distribution of positive
charge so that the attack by nucleophiles may take place at the
electrophilic carbon atoms as well.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
We are grateful to Mr. Jirı
̌
Kosina for HRMS measurements.
ABBREVIATIONS
■
C2-PhNHA, 2-phenylaminoadenine; C8-PhNHA, 8-phenyl-
aminoadenine; C8-PhNHdA, 8-phenylamino-2′-deoxyadeno-
sine; C8-PhNHG, 8-phenylaminoguanine; C8-PhNHdG,
8-phenylamino-2′-deoxyguanosine; dA, 2′-deoxyadenosine;
dG, 2′-deoxyguanosine; DMF-G, N2-(dimethylaminomethy-
lene)guanine; EDTA, ethylendiaminetetraacetic acid; ESI,
electrospray ionization; N7-2APA, 7-(2-aminophenyl)adenine;
N7-4APA, 7-(4-aminophenyl)adenine; N7-2APdG, 7-(2-ami-
nophenyl)-2′-deoxyguanosine; N7-4APdG, 7-(4-aminophenyl)-
2′-deoxyguanosine; N7-2APG, 7-(2-aminophenyl)guanine;
N7-4APG, 7-(4-aminophenyl)guanine; N6-2APA, N6-(2-
aminophenyl)adenine; N6-4APA, N6-(4-aminophenyl)adenine;
N2-2APG, N2-(2-aminophenyl)guanine; N2-4APG, N2-(4-
aminophenyl)guanine; N7-2NPDG, 7-(2-nitrophenyl)-N2-
(dimethylaminomethylene)guanine; N7-4NPDG, 7-(4-nitro-
phenyl)-N2-(dimethylaminomethylene)guanine; N7-2NPG, 7-
(2-nitrophenyl)guanine; N7-4NPG, 7-(4-nitrophenyl)guanine;
2-NPBA, 2-nitrophenylboronic acid; 4-NPBA, 4-nitrophenyl-
boronic acid; PhNH+, phenylnitrenium; PhNHOAc, N-aceto-
xyaniline; PhNHOH, phenylhydroxylamine; TBD, 1,5,7-
triazabicyclo[4.4.0]dec-5-ene; TEA, triethylamine; TMEDA,
N,N,N′,N′-tetramethylethylenediamine; TRIS, tris(hydro-
xymethyl)aminomethane
Unlike many arylnitrenium ions derived from carcinogenic
arylamines, PhNH+ has a very short lifetime of 125−240 ps in
aqueous solutions.23 Even a lower lifetime of 110 ps was
reported recently in a study describing the first spectroscopic
observation of this species in 100% formic acid.26 Nonetheless,
our results show that despite its short lifetime, PhNH+ is able to
react rather selectively, if not effectively, with both dG and dA.
Theoretically, adducts can be formed through two different
electrophilic carbon atoms of phenylnitrenium, i.e., C2 and C4.
However, only the latter type of adducts has been detected in
our study.
CONCLUSIONS
■
The phenylnitrenium ion generated from PhNHOAc can react
under physiological conditions with dA and dG as well as with
nucleophilic sites in the DNA through both the charged
nitrogen and the C4 position. Identification of N7-4APG,
which was found as a minor DNA adduct formed by these
reactions, represents the first evidence of arylnitrenium adduct
formation at the N7 position of dG, which is known to be a
prominent site of attack by other electrophilic species. Further
studies are needed to learn whether significant amounts of
N7-guanine adducts are formed from arylnitrenium ions
derived from strongly mutagenic arylamines and/or nitro-
aromatics with long lifetime in aqueous solutions and to
determine their biological significance.
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: +420 220 444 165. E-mail: linharti@vscht.cz.
Funding
This study was funded by institutional sources of the University
of Chemistry and Technology, Prague through the program of
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