Synthesis of C8-Arylamine-Modified 2′-Deoxyadenosine Phosphoramidites and their Site-Specific Incorporation into Oligonucleotides

Article abstract of DOI:10.1002/cbic.201100573

Adducts of C8-(N-acetyl)-arylamines and 2′-deoxyadenosine were synthesised by palladium-catalysed C-N cross-coupling chemistry. These 2′-dA adducts were converted into the corresponding 3′-phosphoramidites and site-specifically incorporated into DNA oligonucleotides, which were characterised by mass spectrometry, UV thermal-stability assays and circular dichroism. These modified oligonucleotides were also used in EcoRI restriction assays and in primer-extension studies with three different DNA polymerases. The incorporation of the 2′-dA lesion close to the EcoRI restriction site dramatically reduced the susceptibility of the DNA strand to cleavage; this indicates a significant local distortion of the DNA double helix. The incorporation of the acetylated C8-2′-dA-phosphoramidites into 20-mer oligonucleotides failed, however, because the N-acetyl group was lost during the deprotection process. Instead the corresponding C8-NH-2′-dA-modified oligonucleotides were obtained. The effect of the C8-NH-arylamine-dA lesion on the replication by DNA polymerases was clearly dependent both on the polymerase used and on the arylamine-dA damage.

Full text of DOI:10.1002/cbic.201100573

DOI: 10.1002/cbic.201100573
Synthesis of C8-Arylamine-Modified 2’-Deoxyadenosine
Phosphoramidites and their Site-Specific Incorporation
into Oligonucleotides
Zita Szombati,[a] Sabrina Baerns,[b] Andreas Marx,[b] and Chris Meier*[a]
Adducts of C8-(N-acetyl)-arylamines and 2’-deoxyadenosine
were synthesised by palladium-catalysed CꢀN cross-coupling
chemistry. These 2’-dA adducts were converted into the corre-
sponding 3’-phosphoramidites and site-specifically incorporat-
ed into DNA oligonucleotides, which were characterised by
mass spectrometry, UV thermal-stability assays and circular di-
chroism. These modified oligonucleotides were also used in
EcoRI restriction assays and in primer-extension studies with
three different DNA polymerases. The incorporation of the 2’-
dA lesion close to the EcoRI restriction site dramatically re-
duced the susceptibility of the DNA strand to cleavage; this in-
dicates a significant local distortion of the DNA double helix.
The incorporation of the acetylated C8-2’-dA-phosphoramidites
into 20-mer oligonucleotides failed, however, because the N-
acetyl group was lost during the deprotection process. Instead
the corresponding C8-NH-2’-dA-modified oligonucleotides
were obtained. The effect of the C8-NH-arylamine-dA lesion on
the replication by DNA polymerases was clearly dependent
both on the polymerase used and on the arylamine-dA
damage.
Introduction
Damage to DNA can result from several events such as irradia-
tion (TT-photodimers), oxidation (8-oxo-G damage) and the
action of chemical reagents (alkylating agents). These different
forms of damage can have severe consequences for the bio-
logical system if they are not repaired by, for example, the
base excision repair (BER) or nucleotide excision repair (NER)
pathways. Such damage can lead to mutations and sometimes
these mutations can even lead to the induction of carcinogen-
esis.
mic acids 5. Further esterification again leads to highly reactive
N-acetoxy compounds 6.
Solvolysis of compounds 6 generates the corresponding
highly reactive N-arylnitrenium ions 4. The predominant reac-
tions of the arylnitrenium ions 4 occur at the C8-positions of
2’-deoxyguanosine (dG) and 2’-deoxyadenosine (dA), leading
to the corresponding N-H or N-Ac-C8 adducts 7a and 7b
(Scheme 1). Moreover, N2-dG- and N6-dA adducts have been
identified as minor products.[4,5]
Poly- and monocyclic aromatic amines belong to the class of
chemical carcinogens. The covalent chemical modification of
the double helix seems to play a significant role in the poten-
tial induction of chemical carcinogenesis by aromatic amines
of type 1 (Scheme 1).[1] Aromatic amines are not toxic per se,
however. The so-called ultimate carcinogens 6 are formed
through metabolic processes.
Marroquin and Coyote reported that the binding preferen-
ces of N-hydroxy-N-acetylaminofluorene (AAF) for poly(G) and
for poly(A) strands were about 6 to 1.[5] In their studies, two
products were found in the case of 2’-dA. N6-AAF-dA was iden-
tified as the major product and the C8-AAF-dA adduct as
a minor product.[6]
A first report on N-acetylated adducts concerned the corre-
sponding C8-dG adduct. Zhou and Romano reported on the
synthesis of N-acetylaminofluorene-2’-dG adducts and their
conversion into phosphoramidites. Standard ammonia depro-
tection after the oligonucleotide synthesis failed because of
the presence of the base-labile N-acetyl group, so the more
Oxidation reactions are involved in the conversion of the ar-
omatic amines into more hydrophilic compounds. One option
involves the oxidation of the aromatic residues to afford
phenol derivatives. The hydroxy groups are then esterified to
give glucuronic esters. These esters enter into the detoxifica-
tion pathway. However, cytochrome P450-catalysed oxidation
of the amino groups leads to the corresponding N-hydroxyaryl-
amines 2,[2,3] which, after esterification by N-acetyltransferases
(NATs) or transformation into sulfates by sulfotransferases (STs),
form N-acetoxy- or N-sulfatoarylamines (e.g., 6). Through this
process highly electrophilic metabolites are generated, and
these are believed to be the ultimate carcinogen.
[a] Dr. Z. Szombati, Prof. Dr. C. Meier
Organic Chemistry, Department of Chemistry
Faculty of Sciences, University of Hamburg
Martin-Luther-King-Platz 6, 20146 Hamburg (Germany)
E-mail: chris.meier@chemie.uni-hamburg.de
[b] S. Baerns, Prof. Dr. A. Marx
Department of Chemistry and
Konstanz Research School of Chemical Biology
University of Konstanz
N-Acetylated N-arylamines 3 might also play an important
role. These can be formed from the aromatic amines 1 by en-
zymatic acetylation. Oxidation can then generate arylhydroxa-
Universitꢀtsstrasse 10, 78457 Konstanz (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100573.
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DNA Damage by Aromatic Amines
hydrogen bond stabilisation in the double helix.[11,12]
It was shown that C8-NAc-arylamine-dG damage in
the DNA often caused frameshift mutations.[13]
In 2006 we published a first short report on the
chemical synthesis of C8-NH-arylamine-2’-dA adducts
[(NH-aa)-dA], their phosphoramidites and their site-
specific incorporation into oligonucleotides.[14] In
2007 we reported a simple and efficient route to 8-
(N-acetylarylamine)-2’-dG adducts [(NAc-aa)-dG] and
their phosphoramidites.[15] Until now, however, noth-
ing has been published on the synthesis of C8-(NAc)-
arylamine adducts of 2’-dA. Here, though, we report
on the efficient synthesis of dA adducts of this type,
the preparation of phosphoramidites and their site-
specific incorporation into oligonucleotides of short
mixed sequences. In addition to CD and Tm measure-
ments, the effects of these adducts on the cleavage
of a damaged DNA duplex by the endonuclease
EcoRI was investigated. Moreover, template 20-mer
DNA oligonucleotides containing these lesions were
prepared and studied in relation to primer extension
by three different DNA polymerases.
Results and Discussion
Synthesis of 8-(N-acetyl)-arylamine-2’-deoxy-
adenosine [8-(NAc-aa)-dA] adducts
It was first attempted to introduce the N-acetylated
aromatic amines directly through the Pd-based cross-
coupling chemistry that had been used previous-
ly.[9,14,16] This failed, however, and only the C8-NH-dA
adduct was formed, although in low chemical yields.
Therefore, a linear synthesis was used and the reac-
Scheme 1. Metabolism of aromatic amines, together with isolated and identified C8-aryl-
amine-purine nucleoside adducts (NAT: N-acetyltransferase; CYP: cytochro-
me P450).
base-labile fluorenylmethoxycarbonyl (Fmoc) group was used
as protecting group.[7,8]
Later, Gillet and Schꢁrer introduced a new strategy for the
introduction of the N-acetyl group, the subsequent synthesis
of the N-acetylated dG-phosphoramidites and their site-specific
incorporation into DNA oligonucleotides. They removed the
protecting groups by overnight treatment with diisopropyla-
mine (DIPA), because the loss of the N-acetyl group was ob-
served on application of ammonia.[9,10]
Acetylated and non-acetylated adducts can cause different
extents of local distortion in the DNA helix, due to significant
Scheme 2. anti and syn conformations of the non-acetylated and the N-ace-
differences in the conformations of the glycosidic bonds in the
C8-modified 2’-dG-nucleosides (Scheme 2).[10,11] Whereas C8-
NH-2’-dG adducts (7a) preferred the anti conformations of
their glycosidic bonds, placing the aromatic moieties in the
major groove of the double helix and so should not in princi-
ple influence the hydrogen bridge stabilisation in the helix, the
N-acetylated C8-2’-dG adducts (8) were found to prefer the syn
conformations, which place the polycyclic aromatic systems
within the double helix and displace the heterocyclic nucleo-
bases from the helix into the major groove, resulting in loss of
tylated adducts.
tion sequence depicted in Scheme 3 was found to be appro-
priate for the synthesis of phosphoramidites 17. Bromination
of 2’-dA (9) at C8[17] and TBDMS protection gave product 11.
Next, Pd-catalysed Buchwald–Hartwig cross-coupling led to C8-
NH-aa-dA adducts 12a–e. The exocyclic amino group was then
doubly protected to give compounds 13, which were used for
acetylation. Best results were obtained when 4-DMAP (3 equiv)
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701

C. Meier et al.
and Ac2O (3 equiv) in anhydrous pyridine were used.
The reactions took 30–72 h, during which one of the
benzoyl groups was also cleaved to give compounds
14a–e. Adducts 14a–e each showed a typical line
1
broadening in the H NMR spectra at 298 K, due to
the amide bond resonance of the benzoyl group at
the exocyclic amino group. Beland reported the
same for the acetylated 2’-dG adducts.[18] However,
when the spectra were recorded at 328 K in
[D6]DMSO, the line broadening was significantly less.
Because of the introduced N-acetyl group, the re-
moval of the TBDMS groups had to be done under
mild acid conditions. The use of tetra-n-butylammo-
nium fluoride led to loss of the N-acetyl groups.
However, with buffering of the TBAF cleavage solu-
tion with acetic acid, the TBDMS groups were
cleaved without loss of the N-acetyl groups and gave
compounds 15. The reactions with the N-acetylated
2’-dA derivatives were four times slower than reac-
tions performed with 2’-dG.[9,10] Deprotection was
also possible with Et3N·3HF, although the workup
was found to be quite difficult.
The 5’-positions were then protected with DMTr
groups and finally compounds 16 were converted
into the corresponding phosphoramidites 17
(Scheme 3). In both cases, column chromatography
was performed on Al2O3 (act. III).
In the synthesis summarised in Scheme 3, both C8-
NH-2’-dA adducts and C8-NAc-2’-dA adducts were
prepared, and so the syn or anti preferences of the
glycosidic bonds in the 2’-dA adducts were studied
by NOESY-NMR spectroscopy. Figure 1A shows an
observed cross-peak between the H1’ proton of the
sugar and the H2 proton of the nucleobase that can
be correlated to an anti orientation of the nucleo-
base. In contrast, cross-peaks between H2 and the 5’
protons or the 5’ hydroxy proton typical of a syn con-
formation were absent. Figure 1B, however, shows
an observed cross-peak between H2 and the 5’-OH
group, which indicated a preferred syn conformation,
whereas the H2 proton showed no cross-peak with
H1’.
Site-specific synthesis of oligonucleotides contain-
ing C8-(NAc-aa)-2’-dA adducts of different aromatic
amines
Scheme 3. Acetylation route and synthesis of the corresponding phosphoramidites 17.
a) Br2, NaOAc buffer (pH 5.0), RT, 16 h, 83%; b) TBDMSCl, imidazole, pyridine, RT, 16 h,
92%; c) aryl-NH2, 30 mol% rac-BINAP, 10 mol% Pd2(dba)3, Cs2CO3, 1,2-DME, 958C, 24–48 h,
88% a (Ar=phenyl), 98% b (=3,5-dimethylphenyl, 98% c (=4-methoxyphenyl), 90% d
(=4-biphenyl), 91% e (=2-fluorenyl); d) BzCl, pyridine, RT, 16 h, 86% (a), 96% (b), 90%
For the incorporation of the dA adducts into oligonu-
cleotides, phosphoramidites 17a–e were dissolved in
acetonitrile (0.1m solutions), and their coupling steps
were repeated three times, with total coupling effi-
(c), 95% (d), 82% (e); e) 4-DMAP, Ac2O, pyridine, RT, 72 h, 81% (a), 43% (b), 81% (c), 63% ciencies of >90%. In this way, three different site-
(d), 80% (e); f) i: Et3N, Et3N·3HF, CH2Cl2/THF, ii: AcOH, TBAF, THF, RT, 24–48 h, 93% (a),
90% (b) 90% (c) 93% (d) 91% (e); g) DMTrCl, AgNO3, pyridine, 3–16 h, RT, 52% (a), 67%
(b), 62% (c), 66% (d), 70% (e); h) bis-N,N’-diisopropylamino-(2-O-cyanoethyl)-phosphite,
DCI, CH2Cl2, CH3CN, 24 h, RT, 66% (a), 55% (b), 63% (c), 66% (d), 66% (e).
specifically modified oligonucleotide sequences were
prepared: the NarI oligonucleotide [5’-d(CTCGGC
ACCATC)-3’], a palindromic 12-mer EcoRI oligonucleo-
tide [5’-d(GTAGAA TTCTAC)-3’] and a 20-mer oligonu-
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DNA Damage by Aromatic Amines
Figure 2. HPLC chromatogram of the NarI sequence containing a C8-(NAc-
phenyl)-2’-dA lesion (18c) after treatment with DIPA. Peaks in lanes 1 and 3
with retention times of 29.84 and 29.92 min correspond to the target oligo-
nucleotide 18c whereas the peaks at 31.69 and 31.60 min correspond to the
N-deacetylated C8-(NH-phenyl)-2’-dA damaged oligonucleotide (18b). Elu-
tion conditions: CH3CN gradient 0–23% in TEAA buffer (pH 6.9) over 50 min.
tive side effect because both the C8-(NH-aa)-dA- and the C8-
(NAc-aa)-dA-modified oligonucleotides were obtained. Finally,
5% DIPA was used for deprotection/cleavage in all cases. In
these reactions b-mercaptoethanol was added to prevent oxi-
dative rearrangement in the presence of strong bases and
oxygen, as observed previously for oligonucleotides containing
the C8-NH-aminofluorene-2’-dG adduct.[19,20] The obtained oli-
gonucleotides were purified by reversed-phase HPLC and char-
acterised by MALDI-TOF or ESI mass spectrometry (negative
mode).
However, the isolation of the C8-(NAc-aa)-dA-containing NarI
and EcoRI sequences was successful. In contrast, although fur-
ther deprotection methods were unsuccessfully tested, none
of the N-acetylated, modified 20-mer oligonucleotides could
be isolated, but the C8-NH-arylamine-modified 20-mer oligonu-
cleotides were obtained.
Figure 1. NOESY-NMR spectra of A) compound 12a, and B) compound 15a
in [D6]DMSO at 328 K.
cleotide [5’-d(ACATAA GCATCT ACGACG CG)-3’] needed for
DNA polymerase primer extension assays.
An important matter after the synthesis of the oligonucleo-
tides was to find appropriate deprotection conditions because
these adducts, particularly the N-acetyl group, are known to
be base-labile. Standard deprotection with ammonia (4 h,
558C) was tried first, but failed; only the non-acetylated C8-
NH-2’-dA arylamine-modified oligonucleotide was isolated.
Several variations of the ammonia treatment (3!24 h, RT!
558C) also failed. Finally, diisopropylamine (DIPA) in methanol
was used both for the deprotection and for cleavage from the
support. Two different concentrations (5 and 10% DIPA) were
tried, at room temperature and at 558C. Figure 2 shows at-
tempts at deprotection with the C8-(NAc-phenyl)-2’-dA oligo-
nucleotide 18c. Clearly, at room temperature no oligonucleo-
tide was detectable either with 5 or 10% DIPA (lanes 2 and 4;
Figure 2). On overnight treatment (14 h) with DIPA (5 or 10%)
in MeOH at 558C, however, successful deprotection/cleavage
was observed (lanes 1 and 3), but the acetylated (18c;
29.9 min) and non-acetylated (18b; 31.6 min) oligonucleotides
were obtained in comparable amounts. Although this was un-
expected, due to the results reported previously,[9] it was a posi-
Melting temperature (Tm) and circular dichroism studies
All oligonucleotides were hybridised with the complementary
strands and the thermal stabilities of the double strands of the
C8-(NAc-aa)-dA adducts relative to those of the C8-NH-aa-dA
adducts were measured by UV melting temperature analysis.
The data for the NarI oligonucleotides 18a–i are given in
Table 1.
For the C8-NH-phenyl- and C8-NH-4-methoxyphenyl-contain-
ing damaged 2’-dA oligonucleotides 18b and 18d, decreases
of ꢁ88C (relative to the Tm value of the unmodified NarI oligo-
nucleotide 18a, Tm =58.28C) were observed. Interestingly, for
the oligonucleotide bearing the C8-NH-biphenyl-2’-dA lesion
18h, no further significant decrease in thermal stability was
observed. In the case of the 3,5-dimethylphenyl lesion 18 f,
however, a decrease of ꢁ158C was observed. Here, the influ-
ence of the two methyl groups in the 3,5-dimethylphenyl
system on the thermal stability is more important than the
second aromatic ring in the biphenyl residue. In the case of
the N-acetylated lesions, further decreases in thermal stability
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C. Meier et al.
almost no differences between the NH- and NAc-arylamine-dA
lesions were found. Here the destabilisation is more pro-
nounced because the hybridisation at the end of a duplex is
less stable, and this effect is attenuated if an additional struc-
tural distortion is present. The hybridisation data for the 20-
mer oligonucleotides 20a–j are summarised in Table 3.
Table 1. Tm values of the duplexes formed with NarI oligonucleotides
18a–i.
Oligonucleotide
Tm [8C]
5’-d(CTC GGC ACC ATC)-3’ (18a)
5’-d(CTC GGC A(phenyl)CC ATC)-3’ (18b)
58.2
50.1
48.2
51.0
47.3
45.1
42.1
49.0
45.8
5’-d(CTC GGC A(Ac-phenyl)CC ATC)-3’ (18c)
5’-d(CTC GGC A(4-methoxyphenyl)CC ATC)-3’ (18d)
5’-d(CTC GGC A(Ac-4-methoxyphenyl)CC ATC)-3’ (18e)
5’-d(CTC GGC A(3,5-dimethylphenyl)CC ATC)-3’ (18 f)
5’-d(CTC GGC A(Ac-3,5-dimethylphenyl)CC ATC)-3’ (18g)
5’-d(CTC GGC A(biphenyl)CC ATC)-3’ (18h)
5’-d(CTC GGC A(Ac-biphenyl)CC ATC)-3’ (18i)
Table 3. Tm values of the duplexes formed with 20-mer oligonucleotides
20a–j.
Oligonucleotide
Tm [8C]
5’-d(ACA TAA GCA TCT ACG ACG CG)-3’ (20a)
5’-d(ACA TA(phenyl)A GCA TCT ACG ACG CG)-3’ (20b)
5’-d(ACA TA(4-methoxyphenyl)A GCA TCT ACG ACG CG)-3’ (20d) 60.8
5’-d(ACA TA(3,5-dimethylphenyl)A GCA TCT ACG ACG CG)-3’ (20 f) 57.8
64.8
61.1
Conditions: NarI oligonucleotide (1 nmol), complementary oligonucleo-
tide strand (1 nmol), phosphate buffer (pH 7.2, 10 mm), NaCl (140 mm),
EDTA (1.0 mm).
5’-d(ACA TA(biphenyl)A GCA TCT ACG ACG CG)-3’ (20h)
5’-d(ACA TA(fluorenyl)A GCA TCT ACG ACG CG)-3’ (20j)
58.7
61.6
Conditions: 20-mer oligonucleotide (1 nmol), complementary oligonu-
cleotide strand (1 nmol), phosphate buffer (pH 7.2, 10 mm), NaCl
(140 mm), EDTA (1.0 mm).
of ꢁ2–48C relative to their non-acetylated counterparts was
determined.
For the palindromic EcoRI oligonucleotides, the effect on the
Tm values was expected to be more pronounced, because after
duplex formation a lesion is present in each strand. Two differ-
ent oligonucleotide modifications differing in the positions of
the lesions were prepared. In one set of oligonucleotides the
lesions were at the cleavage position in the restriction region
(fifth base from the 5’-end) whereas the other set of oligonu-
cleotide had the lesions only one base before the cleavage site
(third base from the 5’-end). The Tm values are summarised in
Table 2.
The double strand of the unmodified reference oligonucleo-
tide showed a thermal stability of about 658C. The N-phenyl,
the N-(4-methoxyphenyl) and the N-fluorenyl-NH adducts de-
creased the Tm value by ꢁ3–48C. The strongest destabilisation
was again found for the 3,5-dimethylaniline lesion—20 f—fol-
lowed by the biphenylamine residue in 20h.
In addition, the circular dichroism (CD) spectra of all synthes-
ised oligonucleotides as hybrids with the complementary
strands were recorded to verify the overall B-type DNA confor-
mations of the lesion-containing DNA hybrids and the unmodi-
fied reference duplex. In the case of the NarI sequence 18a–
i no conformational difference between the reference oligonu-
cleotide 18a and the oligonucleotides containing C8-NAc-aa-
and C8-NH-aa-dA was observed, because in each case a positive
Cotton effect was observed at 273 nm together with a nega-
tive, less pronounced one at 240 nm typically found for B-type
DNA. However, the CD curves were less shaped, which indi-
cates less structured helixes. In addition, no overall conforma-
tional differences were observed in the cases either of the
EcoRI oligonucleotides (Figure 3) of the 20-mer duplexes.
Table 2. Duplex Tm values of the EcoRI oligonucleotides 19a–m.
Oligonucleotide
Tm [8C]
5’-d(GTA GAA TTC TAC)-3’ (19a)
5’-d(GTA GA(phenyl)A TTC TAC)-3’ (19b)
41.7
41.3
41.5
32.6
34.3
42.8
40.2
30.1
31.5
29.1
32.0
33.4
33.6
5’-d(GTA GA(Ac-phenyl)A TTC TAC)-3’ (19c)
5’-d(GTA GA(4-methoxyphenyl)A TTC TAC)-3’ (19d)
5’-d(GTA GA(Ac-4-methoxyphenyl)A TTC TAC)-3’ (19e)
5’-d(GTA GA(biphenyl)A TTC TAC)-3’ (19 f)
5’-d(GTA GA(Ac-biphenyl)A TTC TAC)-3’ (19g)
5’-d(GTA(phenyl) GAA TTC TAC)-3’ (19h)
5’-d(GTA(Ac-phenyl) GAA TTC TAC)-3’ (19i)
5’-d(GTA(4-methoxyphenyl) GAA TTC TAC)-3’ (19j)
5’-d(GTA(Ac-4-methoxyphenyl) GAA TTC TAC)-3’ (19k)
5’-d(GTA(biphenyl) GAA TTC TAC)-3’ (19l)
5’-d(GTA(Ac-biphenyl) GAA TTC TAC)-3’ (19m)
Conditions: EcoRI oligonucleotide (2 nmol), phosphate buffer (pH 7.2,
10 mm), NaCl (140 mm), EDTA (1.0 mm).
Surprisingly, there was almost no thermal destabilisation (rel-
ative to the unmodified reference oligonucleotide 19a) when
the lesion was introduced in the cleavage site, except in cases
involving the 4-methoxyphenyl residue (19d, 19e). Moreover,
there was no effect of acetylation at the nitrogen atom on the
duplex stability. Significant decreases in thermal stability were
observed, however, when the lesions were introduced closer
to the 5’-ends of the oligonucleotides (19h–m). As above,
Figure 3. CD spectra of EcoRI oligonucleotides 19a–c.
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DNA Damage by Aromatic Amines
EcoRI restriction assay
To investigate the effects of arylamine-dA-modified oligonucle-
otides on endonuclease restriction, an EcoRI restriction assay
was performed.[16] The enzyme EcoRI cleaves both strands of
a DNA double helix. We have shown before that, under the
reaction conditions used here, the enzyme EcoRI cleaved the
self-complementary, undamaged 12-mer oligonucleotide 19a
[5’-d(GTA GAA TTC TAC)-3’] into a tetramer (GTAG) and an octa-
mer strand (AATTCTAC).[16] For oligonucleotide 19a, a half-life
of 2.45 h was determined (270 units of enzyme, DTT buffer at
238C). The half-life was calculated as described previously.[21]
When the same restriction assay was performed with oligonu-
cleotides 19b–f under identical experimental conditions, no
cleavage of any of these lesion-containing oligonucleotides
was detected. As an example, the experiment with 19e fol-
lowed over 24 h is illustrated in Figure 4.
Figure 5. HPLC chromatogram for the EcoRI restriction assay with 19h.
Table 4. Cleavage of the damaged oligonucleotides 19 by the restriction
endonuclease EcoRI.
Oligonucleotide
t1/2
5’-GTAGAATTCTAC-3’ (19a)
2.5
5’-d(GTA GA(phenyl)A TTC TAC)-3’ (19b)
no cleavage
no cleavage
no cleavage
no cleavage
no cleavage
no cleavage
3.3
5’-d(GTA GA(Ac-phenyl)A TTC TAC)-3’ (19c)
5’-d(GTA GA(4-methoxyphenyl)A TTC TAC)-3’ (19d)
5’-d(GTA GA(Ac-4-methoxyphenyl)A TTC TAC)-3’ (19e)
5’-d(GTA GA(biphenyl)A TTC TAC)-3’ (19 f)
5’-d(GTA GA(Ac-biphenyl)A TTC TAC)-3’ (19g)
5’-d(GTA(phenyl) GAA TTC TAC)-3’ (19h)
5’-d(GTA(Ac-phenyl) GAA TTC TAC)-3’ (19i)
5’-d(GTA(4-methoxyphenyl) GAA TTC TAC)-3’ (19j)
5’-d(GTA(Ac-4-methoxyphenyl) GAA TTC TAC)-3’ (19k)
5’-d(GTA(biphenyl) GAA TTC TAC)-3’ (19l)
5’-d(GTA(Ac-biphenyl) GAA TTC TAC)-3’ (19m)
inhibition
3.3
inhibition
3.1
inhibition
Figure 4. HPLC chromatogram for the EcoRI restriction assay with 19e.
Primer-extension assay
Clearly, the presence of the C8-(NH-aa)- or C8-(NAc-aa)-dA
damage in the cleavage site caused severe conformational dis-
tortion and steric blocking, thus making the enzyme unable to
bind and/or to cleave the DNA double strand. However, with
a shift of the modification site only one position away from
the cleavage site, restriction was observed but at lower rates
(Figure 5). For the C8-NH-aa-dA damaged oligonucleotides
half-lives of 3.3 h were calculated in the cases of aniline and 4-
methoxyaniline, whereas the polycyclic 4-aminobiphenyl
damage led to a half-life of 2.9 h. In the case of the N-acetylat-
ed modification 19m the EcoRI restriction assay showed a sur-
prising effect; it seemed that the slow enzymatic reaction even
stopped after 6 h, with no change being observable after 9 h
of incubation relative to the values obtained after 6 h. Addition
of fresh enzyme after 9 h led to further cleavage, which again
stopped after several hours. The same effect was observed in
the case of the N-acetylated monocyclic lesions 19i and 19k.
This behaviour was not seen with the corresponding oligonu-
cleotides containing the C8-(NH-aa)-dA lesions. These results
suggest inhibition of the enzyme by an unknown mechanism.
Table 4 summarises the calculated half-lives, all calculated with
the assumption of first-order reactions.
As investigations of other DNA adducts have shown, covalent
DNA modifications significantly hamper the selectivity and effi-
ciency of DNA synthesis by replicative DNA polymerases,
whereas other DNA polymerases are effective in performing
lesion bypass beyond the site of damage.[22] Here, three DNA
polymerases from different DNA polymerase families were
studied with regard to their effectiveness in bypassing the C8-
NH-arylamine-dA lesions (Figure 6).
The experiments were performed under standing-start con-
ditions with a 32P-labelled primer/template complex (Figure 6).
Single incorporations were examined in order to gain insights
into the impact of the lesion on selectivity, whereas additional
experiments with all four dNTPs were used to study lesion
bypass capability.
Human DNA polymerase b, a member of the DNA polymera-
se X family involved in DNA repair, was investigated first.[23] In-
corporation opposite the DNA lesion was successful and the
canonical dTTP was always preferably chosen. In the case of
the 4-aminobiphenyl-dA lesion, however, misinsertion of the
non-canonical dGMP was also observed to some extent. In the
presence of all four dNTPs, complete elongation was observed
in the reference experiment, as expected. Interestingly, in the
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705

C. Meier et al.
These experiments established
that the lesions have marked
influences on high-fidelity poly-
merases due to the structural
changes at the damaged sites.
At the very least, considerable
reductions in the elongation effi-
ciencies were always detected.
A few differences from our
previous study based on C8-NH-
arylamine adducts of 2’-deoxy-
guanosine[16] were identified. In
the cases of 4-aminobiphenyl
and anisidine, DNA polymerase b
incorporated only the canonical
dC and full elongation of the
primer was observed. With Dpo4
DNA polymerase, however, de-
spite the selective incorporation
of dC, a pronounced slowing of
the replication was observed
after the dC incorporation. Final-
ly, Pfu DNA polymerase led to
significant misincorporation op-
posite the dG lesion. In addition
to dC, dA and dT were also
found to be incorporated by the
enzyme and again a pronounced
slowing after the elongation was
observed.[16] Interestingly, the dA
Figure 6. C8-Arylamine-2’-dA lesions and DNA replication.
and the dG lesions caused differ-
ent misincorporations in differ-
ent amounts. However, in the
case of the lesion-containing oligonucleotides, products two
nucleotides shorter were also observed beside the full-length
products. In the case of the 2-aminofluorene-dA damage this
was even the predominant replication product. The DNA syn-
thesis proceeded with less efficiency than in the case of the
unmodified reference.
cases of misincorporation, the dA lesion led to the introduction
of dG instead of dT whereas in the case of the dG lesion dA
was incorporated instead of dC. The consequence is that in
both lesions a pair of purine nucleosides is produced at the
site of DNA damage.
Next, the Sulfolobus solfataricus P2 DNA polymerase IV
(Dpo4), which often serves as a functional and structural model
for Y-family DNA polymerases, was studied.[24] Again, the can-
onical dT was incorporated in all cases of single-incorporation
experiments and again the non-canonical dGTP was added op-
posite the 4-aminobiphenyl lesion. Unlike the first DNA poly-
merase, Dpo4 was effective in bypassing all the lesions when
all four nucleotides were employed, although again with re-
duced efficiency.
Conclusion
A procedure for the synthesis of 8-(N-acetyl)-arylamine-dA
phosphoramidites has been developed. The C8-arylamine moi-
eties were efficiently introduced by using Pd cross-coupling
chemistry. The corresponding phosphoramidites were site-spe-
cifically incorporated into two different 12-mer oligonucleo-
tides. Interestingly, after the automated DNA synthesis, the N-
acetyl-arylamine damaged oligonucleotide and its NH-aryla-
mine counterpart were always isolated, due to the great sensi-
tivity of the N-acetyl group under alkaline conditions. For the
NarI and the self-complementary EcoRI sequences, both the
thermal stabilities and CD spectra were determined. In all
cases, marked decreases in the Tm values were detected for the
lesion-containing DNA strands; this point to marked distortion
of the double helix, resulting in weaker hybridisation between
the two strands. The decreases were even stronger for the N-
acetyl-arylamine damage than for the NH-arylamine lesions.
Finally, the high-fidelity Pyrococcus furiosus (Pfu) DNA poly-
merase (3’!5’ exonuclease-deficient mutant),[25,26] a replicative
DNA polymerase belonging to sequence family B, was investi-
gated. The canonical nucleotide TMP was incorporated pre-
dominantly opposite to the modified dA, but in case of the
C8-NH-4-aminobiphenyl lesion incorporation of the non-canon-
ical dGMP was also observed to some extent. In contrast with
the two other DNA polymerases the incorporation of nucleo-
tides paused immediately after insertion opposite the lesions.
706
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ChemBioChem 2012, 13, 700 – 712

DNA Damage by Aromatic Amines
27.3 mmol) were then added and the reaction mixture was stirred
for 16 h at room temperature. The reaction was stopped by addi-
tion of CH2Cl2. The mixture was washed once with water and once
with brine. The solvent was removed in vacuo. The residue was co-
evaporated with toluene (3ꢂ). The residue was recrystallised from
CH3CN to give 11 as a colourless solid (3.4 g, 7.8 mmol, 85%). M.p.
Whether or not this observation can be correlated with the de-
tected differences in the syn/anti preferences of the glycosidic
bonds in the dA adducts still has to be confirmed. Neverthe-
less, only minimal differences in the CD spectra of the com-
pounds modified with acetylated aromatic amines and those
modified with non-acetylated aromatic amines were measured.
The lesion-containing oligonucleotides were found to be resist-
ant to digestion by EcoRI if the modification was present at
the cleavage site of the EcoRI enzyme. This might indicate
marked local distortion of the DNA hybrid at the modification
site, either blocking the cleavage of the double strand or pre-
venting binding of the restriction enzyme. This effect was inde-
pendent of the C8-NAc-arylamine-dA damage. Previously, NH
adducts of dA or dG had also shown the same behaviour.
However, irrespective of whether a monocyclic or a polycyclic
DNA lesion was investigated, incorporation of the lesion just
one nucleotide prior to the EcoRI cleavage site only slightly re-
duced the half-life of this enzymatic digestion. It thus appears
that the arylamine adducts cause local but not long-ranging
perturbation of helix recognition. Alternatively, just the steric
bulk of the dA adduct in the restriction site might be the
reason for misbinding or failure of enzyme cleavage. Finally, it
was shown that the bypass ability of a lesion in a DNA strand
depended strongly on the DNA polymerase used. In most
cases, the fidelity of nucleotide incorporation opposite the
lesion was not hampered. However, the abilities of the poly-
merases to bypass the lesions were quite different. Only the
strong carcinogen 4-aminobiphenyl showed a different effect
on the replication. With all three polymerases, marked incorpo-
ration of dGTP beside the canonical TTP was detected, leading
to point mutations. The reason for this T!dG misincorpora-
tion is still unknown. Further work to gain insights into the
structural changes in the DNA helix produced by the C8-aryl-
amine adducts is currently underway in our laboratories.
1
1398C; [a]25829 nm =ꢀ8.21 (c=0.67, CHCl3); H NMR (400 MHz, CDCl3):
d=8.25 (s, 1H), 6.34 (t, J=6.7 Hz, 1H), 5.59 (s, 2H), 4.87 (dt, J=5.8,
3.8 Hz, 1H), 3.88–3.97 (m, 2H), 3.62–3.69 (m, 2H), 2.23 (ddd, J=
13.2, 6.9, 4.3 Hz, 1H), 0.93 (s, 9H), 0.82 (s, 9H), 0.14 (s, 6H), ꢀ0.01
(s, 3H), ꢀ0.05 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=154.3,
152.7, 151.2, 128.5, 116.1, 87.9, 86.5, 72.5, 62.8, 36.9, 26.0, 25.8,
18.2, 18.1, ꢀ4.7 ppm; IR (KBr): n˜ =3830, 3182, 2954, 2884, 2856,
1661, 1570, 1492, 1320, 1257, 1110, 838, 776, 669, 597, 554 cmꢀ1
MS (FAB): m/z calcd: 557.18 [M]+; found. 557.12.
;
General Procedure I (GP-I) for the amination of 8-bromo-3’,5’-O-
bis(tert-butyldimethylsilyl)-2’-deoxyadenosine derivatives to
afford compounds 12a–d: Racemic 2,2’-bis(diphenylphosphino)-
1,1’-binaphthyl (rac-BINAP, 30 mol%) and tris(dibenzylidenacetone)-
dipalladium(0) (Pd2(dba)3; 10 mol%) were suspended in anhydrous
1,2-dimethoxyethane (50 mL) and the mixture was stirred for one
hour at room temperature. Compound 11, the amine (2.5 equiv)
and Cs2CO3 (1.5 equiv) were then added and the mixture was
stirred under reflux until the reaction was complete (24–48 h). The
reaction mixture was allowed to cool to room temperature, and
saturated sodium hydrogen carbonate solution (1 mL) was added.
After the addition of brine (10 mL), the layers were separated and
the aqueous layer was extracted with ethyl acetate (3ꢂ10 mL). The
combined organic layers were washed with brine (2ꢂ10 mL) and
with a mixture of brine (10 mL) and water (2 mL). The organic layer
was dried over sodium sulfate and the solvent was removed in
vacuo. Purification of the residue by flash chromatography on
silica gel, with elution with ethyl acetate in petroleum ether (10!
35%), gave the compounds 12.
8-N-Phenyl-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine
(12a): GP-I was used with 11 (3.00 g, 5.38 mmol) and PhNH2, which
afforded the desired product as a pale yellow foam (2.36 g,
4.13 mmol, 77%). M.p. 708C; [a]25809 nm =11.4 (c=0.25, CHCl3);
1H NMR (400 MHz, CDCl3): d=8.19 (s, 1H), 7.87 (s, 1H), 7.57 (d,
J
H,H =8.0 Hz, 2H), 7.34 (t, JH,H =7.7 Hz, 2H), 7.08 (t, JH,H =7.3 Hz, 1H),
Experimental Section
6.45 (dd, JH,H =8.5 Hz, 5.3, 1H), 4.55 (dt, JH,H =5.4 Hz, 2.6, 2.6, 1H),
4.13 (dd, JH,H =5.3, 2.7 Hz, 1H), 3.99 (dd, JH,H =11.7, 2.7 Hz, 1H), 3.87
(dd, JH,H =11.7, 2.8 Hz, 1H), 2.76 (ddd, JH,H =13.2, 8.2, 5.5 Hz, 1H),
2.35 (ddd, JH,H =13.0, 5.4, 2.5 Hz, 1H), 0.93 (s, 9H), 0.79 (s, 9H), 0.13
(s, 3H), 0.12 (s, 3H), ꢀ0.02 (s, 3H), ꢀ0.04 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3): d=151.3, 149.9, 149.2, 139.0, 129.2, 123.4, 120.1,
119.8, 116.2, 88.7, 85.5, 72.1, 63.0, 40.2, 26.1, 25.9, 17.8, 17.7, ꢀ4.4,
ꢀ4.6, ꢀ5.1, ꢀ5.2 ppm; IR (ATR): n˜ =3331, 3184, 2952, 2928, 2885,
2856, 1742, 1636, 1599, 1557, 1498, 1471, 1450, 1346, 1282, 1251,
1108, 1058, 1028, 1006, 951, 883, 831, 775, 747, 690, 668, 577, 538,
521, 498 cmꢀ1; HRMS (ESI+): m/z calcd: 570.3170 [M]+; found:
571.3254 [M+H]+, 593.3066 [M+Na]+, 609.2817 [M+K]+.
General methods: All air- or water-sensitive reactions were per-
formed in flame-dried glassware under nitrogen. Commercial sol-
vents and reagents were used without further purification with the
following exceptions: 1,2-dimethoxy ethane (1,2-DME) was distilled
from potassium under nitrogen, whereas pyridine, dichlorometh-
ane and acetonitrile were distilled from calcium hydride under
nitrogen. Water was purified with a Milli-Q water system. NMR
spectra are reported relative to the corresponding solvent peaks.
1H NMR: 2.50 ppm ([D6]DMSO), 7.26 ppm (CDCl3), 3.31 ppm
(CD3OD) and 7.16 (C6D6). 13C NMR: 39.52 ppm ([D6]DMSO),
77.16 ppm (CDCl3), 49.0 ppm (CD3OD) and 128.06 (C6D6). Thin-layer
chromatography was performed on aluminium sheets coated with
silica gel (Merck, 60 F254). Mass spectra were recorded with VG Ana-
lytical VG/70–250 F (FAB, HR-FAB), Finnigan ThermoQuest MAT
95 XL (ESI, HR-ESI), Agilent Technologies 6224 TOF LC/MS 1200
series (HPLC-ESI-MS) and Bruker BiFlex III (MALDI-TOF) instruments.
8-N-3,5-Dimethylphenyl-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-de-
oxyadenosine (12b): GP-I was conducted with 11 (3.00 g,
5.38 mmol) and 3,5-dimethylaniline, which afforded the desired
product as a pale yellow foam (2.35 g, 3.93 mmol, 73%). M.p.
688C; [a]25809 nm =5 (c=0.25, CHCl3); H NMR (400 MHz, CDCl3): d=
1
8-Bromo-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine
(11): 8-Bromo-2’-deoxyadenosine (10) (3.0 g, 9.1 mmol) was sub-
jected three times to coevaporation of the volatiles with anhydrous
pyridine (5 mL) and then suspended in anhydrous pyridine (30 mL)
under nitrogen. TBDMSCl (4.1 g, 27.3 mmol) and imidazole (1.9 g,
8.15 (s, 1H), 7.90 (brs, 1H), 7.08 (s, 2H), 6.86 (brs, 2H), 8.78 (s, 1H),
6.44 (dd, JH,H =5.4, 8.4 Hz, 1H), 4.55 (dt, JH,H =2.7, 5.6 Hz, 1H), 4.09
(dd, JH,H =2.9, 5.7 Hz, 1H), 3.97 (dd, JH,H =11.6, 3.1 Hz, 1H), 3.85 (dd,
J
H,H =11.6, 3.0 Hz, 1H), 2.78 (ddd, JH,H =13.0, 8.4, 5.9 Hz, 1H), 2.36–
2.27 (m, 1H), 2.32 (s, 6H), 0.93 (s, 9H), 0.79 (s, 9H), 0.13 (s, 3H),
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707

C. Meier et al.
0.11 (s, 3H), ꢀ0.03 (s, 3H), ꢀ0.04 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=150.1, 149.9, 149.0, 140.0, 139.1, 129.0, 119.2, 119.0,
118.7, 88.4, 85.1, 72.2, 62.4, 39.8, 26.0, 25.9, 21.6, 17.8, 17.7, ꢀ4.6,
ꢀ5.7 ppm; IR (ATR): n˜ =3451, 3332, 3186, 2953, 2927, 2856, 1647,
1615, 1595, 1567, 1545, 1462, 1333, 1290, 1253, 1184, 1109, 1072,
1026, 1006, 971, 945, 871, 831, 775, 718, 686, 665, 574, 540, 504,
403 cmꢀ1; HRMS (FAB): m/z calcd: 598.3483 [M]+; found: 599.3562
[M+H]+.
(400 MHz, CDCl3): d=8.59 (s, 1H), 7.68–7.63 (m, 3H), 7.52–7.48 (m,
2H), 7.48–7.38 (m, 3H), 7.40–7.32 (m, 2H), 7.31–7.26 (m, 4H), 7.20–
7.16 (m, 2H), 6.16 (dd, JH,H =6.2, 7.9 Hz, 1H), 4.64–4.56 (m, 1H),
3.97–3.88 (m, 2H), 3.73 (dd, JH,H =9.0, 3.7 Hz, 1H), 3.15–3.06 (m,
1H), 1.75–1.60 (m, 1H), 0.86 (s, 9H), 0.85 (s, 9H), 0.06 (s, 3H), 0.02
(s, 3H), 0.01 (s, 3H), ꢀ0.01 ppm (s, 3H); 13C NMR (101 MHz, CDCl3):
d=172.2, 152.0, 150.5, 149.0, 139.5, 134.5, 132.7, 131.5, 129.7,
129.4, 128.8, 128.7, 128.4, 127.7, 119.0, 118.7, 88.4, 85.3, 72.7, 63.2,
36.7, 26.0, 25.9, 17.8, 17.7, ꢀ4.6, ꢀ5.2 ppm; IR (ATR): n˜ =2952,
2928, 2885, 2855, 1699, 1600, 1575, 1515, 1491, 1471, 1448, 1404,
1350, 1278, 1238, 1177, 1070, 1026, 1003, 984, 933, 909, 834, 775,
753, 715, 692, 617, 602, 579, 506 cmꢀ1; HRMS (ESI+): m/z calcd:
778.3694 [M]+; found: 779.3771 [M+H]+, 801.3584 [M+Na]+,
817.3311 [M+K]+.
8-N-4-Methoxyphenyl-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deox-
yadenosine (12c): GP-I was conducted with 11 (4.00 g, 7.16 mmol)
and 4-methoxyaniline, which afforded the desired product as
22
589 nm
a pale yellow foam (3.86 g, 6.42 mmol, 90%). M.p. 878C; [a]
=
21.0 (c=0.5, CH3Cl); 1H NMR (400 MHz, CDCl3): d=8.15 (s, 1H),
7.84 (brs, 1H), 7.42–7.36 (m, 2H), 6.93–6.86 (m, 2H), 6.47 (dd, JH,H
8.6, 5.5 Hz, 1H), 6.45 (brs, 2H), 4.58–4.52 (m, 1H), 4.08 (dd, JH,H
5.3, 2.7 Hz, 1H), 3.99 (dd, JH,H =11.6, 2.7 Hz, 1H), 3.85 (dd, JH,H
=
=
=
N6-Dibenzoyl-8-N-(3,5-dimethylphenyl)-3’,5’-O-bis(tert-butyldi-
methylsilyl)-2’-deoxyadenosine (13b): GP-II was conducted with
12b (2.34 g, 3.91 mmol), which afforded the product as a yellow
foam (2.81 g, 3.49 mmol, 89%). M.p. 908C; [a]25809 nm =160.6 (c=0.5,
CHCl3); 1H NMR (400 MHz, CDCl3): d=8.58 (s, 1H), 8.12–8.08 (m,
1H), 7.65–7.62 (m, 4H), 7.50–7.36 (m, 6H), 6.91 (s, 1H), 6.80 (s, 2H),
6.13 (dd, JH,H =6.3, 7.8 Hz, 1H), 4.65–4.60 (m, 1H), 3.96–3.90 (m,
2H), 3.87 (dd, JH,H =13.9, 8.6 Hz, 1H), 3.10 (ddd, JH,H =13.2, 7.6,
5.6 Hz, 1H), 2.26 (s, 6H), 1.76–1.68 (m, 1H), 0.87 (s, 9H), 0.85 (s,
9H), 0.07 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H), ꢀ0.01 ppm (s, 3H);
13C NMR (101 MHz, CDCl3): d=172.2, 152.0, 150.5, 149.0, 140.0,
139.4, 134.5, 132.7, 131.4, 130.3, 129.8, 128.7, 128.3, 124.2, 119.0,
118.7, 88.5, 85.1, 72.8, 63.5, 36.4, 26.1, 25.9, 21.2, 17.8, 17.7, ꢀ4.6,
ꢀ4.8 ppm; IR (ATR): n˜ =2952, 2928, 2884, 2856, 1700, 1599, 1575,
1515, 1471, 1462, 1449, 1404, 1350, 1302, 1278, 1238, 1176, 1072,
1027, 1003, 984, 936, 909, 867, 832, 798, 775, 713, 693, 635, 615,
580, 548, 513, 406 cmꢀ1; HRMS (ESI+): m/z calcd: 806.4007 [M]+;
found: 807.4090 [M+H]+.
11.7, 42.8 Hz, 1H), 2.76 (ddd, JH,H =13.0, 8.6, 6.0 Hz, 1H), 2.30 (ddd,
H,H =13.0, 5.5, 2.4 Hz, 1H), 0.93 (s, 9H), 0.79 (s, 9H), 0.13 (s, 3H),
J
0.11 (s, 3H), ꢀ0.03 (s, 3H), ꢀ0.06 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=156.9, 150.8, 150.0, 149.6, 131.4, 123.8, 123.8, 116.8,
114.5, 88.3, 84.9, 72.2, 63.1, 55.7, 39.8, 26.1, 25.9, 18.8, 18.2, ꢀ4.4,
ꢀ4.6, ꢀ5.2, ꢀ5.2 ppm; IR (ATR): n˜ =3332, 3179, 2952, 2928, 2856,
1637, 1604, 1561, 1509, 1463, 1440, 1419, 1390, 1340, 1285, 1245,
1180, 1107, 1059, 1033, 1006, 970, 951, 938, 883, 827, 775, 719,
668, 577, 536, 505, 459 cmꢀ1; MS (FAB): m/z calcd: 600.32 [M]+;
found: 601.33 [M+H]+.
8-N-4-Biphenyl-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyaden-
osine (12d): GP-I was conducted with 11 (3.00 g, 5.38 mmol) and
4-biphenylamine, which afforded the desired product as a pale
yellow foam (2.85 g, 4.41 mmol, 82%). M.p. 858C; [a]25809 nm =33.6
(c=1.1, CHCl3); 1H NMR (400 MHz, CDCl3): d=8.18 (s, 1H), 8.07
(brs, 1H), 7.65–7.61 (m, 2H), 7.61–7.56 (m, 4H), 7.47–7.41 (m, 2H),
7.36–7.30 (m, 1H), 6.46 (dd, JH,H =8.3, 5.5 Hz, 1H), 6.44 (brs, 2H),
4.57 (dt, JH,H =5.7, 2.8 Hz, 1H), 4.11 (dd, JH,H =5.8, 2.9 Hz, 1H), 4.00
(dd, JH,H =11.6, 3.0 Hz, 1H), 3.87 (dd, JH,H =11.6, 3.1 Hz, 1H), 2.82
(ddd, JH,H =13.1, 8.1, 5.9 Hz, 1H), 2.35 (ddd, JH,H =13.0, 5.5, 2.7 Hz,
1H), 0.93 (s, 9H), 0.82 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H), 0.02 (s, 3H),
0.00 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=150.0, 149.7,
149.0, 140.7, 138.0, 136.7, 129.0, 127.8, 127.0, 127.2, 126.9, 120.7,
117.3, 88.4, 85.3, 72.1, 63.0, 40.0, 26.1, 25.9, 18.7, 18.2, ꢀ4.4, ꢀ4.6,
ꢀ5.1, ꢀ5.2 ppm; IR (ATR): n˜ =3345, 2952, 2927, 2884, 2856, 1636,
1598, 1575, 1551, 1487, 1470, 1449, 1411, 1339, 1288, 1252, 1108,
1058, 1025, 1006, 951, 876, 831, 776, 760, 717, 696, 667, 577, 548,
499 cmꢀ1; HRMS (FAB): m/z calcd: 646.3483 [M]+; found: 647.3554
[M+H]+.
N6-Dibenzoyl-8-N-(4-methoxyphenyl)-3’,5’-O-bis(tert-butyldi-
methylsilyl)-2’-deoxyadenosine (13c): GP-II was conducted with
12c (3.84 g, 6.38 mmol), which afforded the product as a yellow
foam (5.06 g, 6.25 mmol, 98%). M.p. 798C; [a]25809 nm =167.8 (c=0.5,
CHCl3); 1H NMR (400 MHz, CDCl3): d=8.59 (s, 1H), 8.13–8.09 (m,
1H), 7.69–7.64 (m, 4H), 7.51–7.37 (m, 6H), 7.13 (d, JH,H =8.8 Hz,
2H), 6.84 (d, JH,H =9.0 Hz, 2H), 6.18 (dd, JH,H =6.6, 7.5 Hz, 1H), 4.66–
4.58 (m, 1H), 3.98–3.88 (m, 2H), 3.80 (s, 3H), 3.73 (dd, JH,H =9.6,
4.2 Hz, 1H), 3.18–3.06 (m, 1H),1.82–1.62 (m, 1H), 0.86 (s, 9H), 0.85
(s, 9H), 0.07 (s, 3H), 0.03 (s, 3H), 0.01 (s, 3H), ꢀ0.01 ppm (s, 3H);
13C NMR (101 MHz, CDCl3): d=172.2, 156.9, 152.8, 151.5, 149.6,
134.5, 132.7, 130.9, 130.3, 129.4, 128.7, 128.6, 128.3, 123.8, 119.0,
118.7, 114.9, 88.4, 85.3, 72.8, 63.3, 55.6, 36.7, 26.1, 25.9, 18.3, 17.6,
ꢀ4.4, ꢀ4.6, ꢀ5.2, ꢀ5.2 ppm; IR (ATR): n˜ =2952, 2928, 2884, 2855,
1694, 1601, 1575, 1509, 1449, 1351, 1280, 1246, 1175, 1108, 1092,
1071, 1026, 984, 934, 910, 866, 832, 775, 693, 664, 641, 606, 580,
549, 528 cmꢀ1; HRMS (FAB): m/z calcd: 808.3800 [M]+; found:
809.3813 [M+H]+.
General Procedure II (GP-II) for the N6-dibenzoylation of the 8-N-
arylamine-2’-dA adducts to afford compounds 13a–d: The ap-
propriate compound 12 was dissolved in anhydrous pyridine
(30 mL) under nitrogen and benzoyl chloride (5 equiv) was added.
The reaction mixture was stirred until the reaction was complete
(12–16 h). It was then diluted with CH2Cl2 (50 mL) and washed with
saturated sodium hydrogencarbonate solution, and the aqueous
layer was extracted twice with CH2Cl2. The organic layer was dried
over sodium sulfate and the solvent was removed in vacuo and co-
evaporation with toluene was performed three times. Purification
of the residue by flash chromatography on silica gel, with elution
with MeOH in CH2Cl2 (0!1%), gave the product 13.
N6-Dibenzoyl-8-N-(4-biphenyl)-3’,5’-O-bis(tert-butyldimethylsilyl)-
2’-deoxyadenosine (13d): GP-II was conducted with 12d (2.84 g,
4.39 mmol), which afforded the product as a yellow foam (3.40 g,
3.98 mmol, 91%). M.p. 1228C; [a]25809 nm =172.2 (c=0.5, CHCl3);
1H NMR (400 MHz, CDCl3): d=8.60 (s, 1H), 7.59–7.50 (m, 6H), 7.49–
7.41 (m, 5H), 7.39–7.34 (m, 1H), 7.32–7.21 (m, 8H), 6.19 (dd, JH,H
6.7, 7.5 Hz, 1H), 4.68–4.58 (m, 1H), 3.99–3.87 (m, 2H), 3.74 (dd,
H,H =9.4, 4.0 Hz, 1H), 3.19–3.08 (m, 1H), 1.80–1.64 (m, 1H), 0.86 (s,
=
J
N6-Dibenzoyl-8-N-(phenyl)-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-
deoxyadenosine (13a): GP-II was conducted with 12a (2.34 g,
4.09 mmol), which afforded the product as a yellow foam (2.82 g,
9H), 0.83 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H), 0.01 (s, 3H), ꢀ0.01 ppm
(s, 3H); 13C NMR (101 MHz, CDCl3): d=172.2, 152.6, 151.8, 150.3,
140.6, 140.1, 139.4, 134.5, 132.8, 131.6, 129.8, 129.0, 128.8, 128.7,
128.4, 128., 127.9, 127.2, 119.0, 88.4, 85.3, 72.7, 63.2, 36.7, 26.1,
1
3.62 mmol, 85%). M.p. 968C; [a]25809 nm =29.2 (c=0.5, CHCl3); H NMR
708
www.chembiochem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 700 – 712

DNA Damage by Aromatic Amines
25.9, 17.8, 17.7, ꢀ4.6, ꢀ5.2 ppm; IR (ATR): n˜ =2952, 2927, 2855,
1700, 1600, 1575, 1517, 1486, 1471, 1448, 1348, 1278, 1248, 1178,
1072, 1026, 1007, 984, 932, 910, 832, 775, 763, 720, 693, 667, 642,
604, 568, 553, 503, 411 cmꢀ1; MS (FAB): m/z calcd: 854.4007 [M]+;
found: 856.4056 [M+2H]+.
3.73 (s, 3H), 3.74–3.69 (m, 1H), 3.57 (ddd, JH,H =11.7, 5.9, 4.8 Hz,
1H), 3.06–2.93 (m, 1H), 2.20 (s, 3H), 2.02–1.91 (m, 1H), 0.87 (s, 9H),
0.85 (s, 9H), 0.07 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H), ꢀ0.01 ppm (s,
3H); 13C NMR (101 MHz, CDCl3): d=172.1, 168.6, 151.4, 150.7,
149.3, 147.5, 139.4, 134.5, 133.8, 133.5, 133.0, 131.3, 128.3, 128.1,
127.8, 121.7, 114.6, 88.3, 84.8, 70.8, 61.8, 55.2, 37.1, 26.1, 25.9, 24.1,
17.8, 17.7, ꢀ4.6, ꢀ4.8 ppm; IR (ATR): n˜ =2953, 2929, 2885, 2856,
2165, 2054, 2037, 2018, 1983, 1951, 1747, 1679, 1610, 1583, 1528,
1509, 1462, 1408, 1374, 1356, 1319, 1283, 1246, 1182, 1108, 1065,
1031, 1000, 950, 891, 831, 803, 774, 719, 692, 661, 608, 580, 550,
526 cmꢀ1; HRMS (ESI+): m/z calcd: 746.3634 [M]+; found: 747.3713
[M+H]+.
General Procedure III for the 8-N-acetyation of the N6-(Bz)2-aryla-
mine-2’-dA adducts to afford compounds 14a–d: The appropri-
ate compound 13 and 4-DMAP (3 equiv) were dissolved in anhy-
drous pyridine (30 mL) under nitrogen and Ac2O (3 equiv) was
added. The reaction mixture was stirred until the reaction was
complete (30–72 h). It was then diluted with dichloromethane
(50 mL) and washed with saturated sodium hydrogencarbonate so-
lution, and the aqueous layer was extracted twice with dichlorome-
thane. The organic layer was dried over sodium sulfate and the sol-
vent was removed in vacuo. Coevaporation three times with tolu-
ene followed. Purification of the residue by flash chromatography
on silica gel, with elution with methanol in dichloromethane (0!
0.5%), gave the desired product.
N6-Benzoyl-8-(N-acetyl)-4-biphenyl-3’,5’-O-bis(tert-butyldimethyl-
silyl)-2’-deoxyadenosine (14d): GP-III was conducted with 13d
(1.63 g, 1.91 mmol), which afforded the desired product as
a yellow foam (1.22 g, 1.53 mmol, 80%). M.p. 658C; [a]25859 nm =164.4
1
(c=0.5, CHCl3); H NMR (400 MHz, CDCl3): d=8.66 (s, 1H), 7.71 (d,
J
H,H =6.6 Hz, 1H), 7.66 (d, JH,H =7.3 Hz, 2H), 7.61 (d, JH,H =7.3 Hz,
2H), 7.49–7.42 (m, 3H), 7.40–7.34 (m, 5H), 6.27 (t, JH,H =7.0 Hz, 1H),
4.78–4.67 (m, 1H), 4.21–3.92 (m, 2H), 3.78–3.69 (m, 1H), 3.43–3.29
(m, 1H), 2.53 (s, 3H), 1.96–1.88 (m, 1H), 0.85 (s, 9H), 0.84 (s, 9H),
0.09 (s, 3H), 0.07 (s, 3H), 0.03 (s, 3H), 0.02 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3): d=172.2, 168.6, 151.6, 149.4, 149.1, 138.9, 138.6,
131.7, 131.5, 131.4, 128.8, 128.3, 128.2, 127.6, 127.4, 126.5, 126.4,
119.0, 118.7, 88.3, 84.9, 70.8, 61.8, 39.0, 26.1, 25.9, 24.1, 17.8, 17.7,
ꢀ4.6, ꢀ4.8 ppm; IR (ATR): n˜ =3033, 2952, 2928, 2884, 2856, 1687,
1612, 1583, 1520, 1486, 1462, 1450, 1434, 1408, 1356, 1317, 1277,
1175, 1109, 1067, 1027, 1006, 949, 833, 776, 762, 710, 695, 666,
555, 505 cmꢀ1; HRMS (FAB): m/z calcd: 792.3851 [M]+; found:
793.3921 [M+H]+.
N6-Benzoyl-8-(N-acetyl)-phenyl-3’,5’-O-bis(tert-butyldimethylsil-
yl)-2’-deoxyadenosine (14a): GP-III was conducted with 13a
(0.71 g, 0.92 mmol), which afforded the desired product as
a yellow foam (0.63 g, 0.88 mmol, 81%). M.p. 878C; [a]25859 nm =12.8
(c=0.5, CHCl3); 1H NMR (400 MHz, CDCl3): d=8.63 (s, 1H), 7.60–
7.55 (m, 2H), 7.49–7.44 (m, 1H), 7.43–7.37 (m, 1H), 7.35–7.30 (m,
2H), 7.30–7.23 (m, 4H), 6.23 (t, JH,H =6.9 Hz, 1H), 4.76–4.68 (m, 1H),
4.01–3.93 (m, 2H), 3.72 (dd, JH,H =14.2, 8.5 Hz, 1H), 3.42–3.28 (m,
1H), 2.53 (s, 3H), 1.97–1.81 (m, 1H), 0.84 (s, 9H), 0.83 (s, 9H), 0.09
(s, 3H), 0.04 (s, 3H), 0.02 (s, 3H), 0.01 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3): d=172.2, 168.7, 152.0, 150.5, 149.0, 139.5, 134.5,
132.7, 131.5, 129.7, 129.4, 128.8, 128.7, 128.4, 127.7, 119.0, 118.7,
88.4, 85.3, 72.7, 63.2, 36.7, 26.0, 25.9, 24.3, 17.8, 17.7, ꢀ4.6,
ꢀ5.2 ppm; IR (ATR): n˜ =2952, 2928, 2885, 2856, 1682, 1609, 1582,
1524, 1493, 1471, 1462, 1406, 1354, 1279, 1250, 1176, 1009, 1066,
1029, 1004, 948, 833, 775, 752, 712, 693, 669, 617, 597, 573, 548,
504 cmꢀ1; HRMS (ESI+): m/z calcd: 716.3538 [M]+; found: 717.3611
[M+H]+.
General procedure IV for the desilylation of the N6-(Bz)-8-(N-ac)-
arylamine-2’-dA adducts to afford compounds 15a–d: The ap-
propriate compound 14 was dissolved in THF (20 mL), and TBAF
(3 equiv, in THF) and AcOH (6 equiv) were then added simultane-
ously. The reaction mixture was stirred until the reaction was com-
plete (48 h). The solvent was removed in vacuo. Purification of the
residue by flash chromatography on silica gel, with elution with
methanol in dichloromethane (0!5%), gave the desired product.
N6-Benzoyl-8-(N-acetyl)-3,5-dimethylphenyl-3’,5’-O-bis(tert-butyl-
dimethylsilyl)-2’-deoxyadenosine (14b): GP-III was conducted
with 13b (2.79 g, 3.46 mmol), which afforded the desired product
N6-Benzoyl-8-(N-acetyl)-phenyl-2’-deoxyadenosine (15a): GP-IV
was conducted with 14a (0.47 g, 0.67 mmol), which afforded the
desired product as a yellow foam (0.27 g, 0.55 mmol, 83%). M.p.
1438C; [a]25859 nm =23.6 (c=0.5, CHCl3/CH3OH 1:1, v/v); 1H NMR
(400 MHz, [D6]DMSO, 323 K): d=10.51 (s, 1H), 8.63 (s, 1H), 7.56 (d,
25
as a yellow foam (1.10 g, 1.48 mmol, 43%). M.p. 828C; [a]
=
589 nm
269.6 (c=0.5, CHCl3); 1H NMR (400 MHz, [D6]DMSO): d=10.68 (s,
1H), 8.61 (s, 1H), 7.64–7.59 (m, 1H), 7.49–7.44 (m, 2H), 7.36–7.32
(m, 2H), 6.93 (s, 1H), 6.88 (s, 2H), 6.21 (t, JH,H =6.5 Hz, 1H), 4.90–
4.56 (m, 1H), 3.95–3.83 (m, 2H), 3.74–3.68 (m, 1H), 3.22–3.03 (m,
1H), 2.19 (s, 6H), 2.16 (s, 3H), 1.97–1.81 (m, 1H), 0.82 (s, 9H), 0.80
(s, 9H), 0.07 (s, 3H), 0.05 (s, 3H), 0.03 (s, 3H), 0.01 ppm (s, 3H);
13C NMR (101 MHz, CDCl3): d=172.2, 168.7, 150.8, 150.1, 149.4,
140.3, 138.7, 131.6, 131.5, 128.9, 128.5, 128.4, 128.2, 127.7, 119.0,
118.7, 88.4, 84.9, 70.9, 61.9, 38.9, 26.0, 25.9, 24.3, 20.6, 17.8, 17.7,
ꢀ4.6, ꢀ5.2 ppm; IR (ATR): n˜ =2952, 2928, 2855, 1691, 1609, 1583,
1525, 1462, 1407, 1353, 1281, 1251, 1176, 1110, 1066, 1029, 1005,
939, 834, 775, 710, 669, 611, 571, 543 cmꢀ1; HRMS (ESI+): m/z
calcd: 744.3851 [M]+; found: 745.3926 [M+H]+.
J
H,H =7.5 Hz, 2H), 7.48–7.44 (m, 1H), 7.40 (t, JH,H =7.8 Hz, 2H), 7.38–
7.34 (m, 2H), 7.33–7.27 (m, 3H), 6.31 (t, JH,H =7.0 Hz, 1H), 5.18 (d,
J
J
H,H =3.7 Hz, 1H), 4.94–4.85 (m, 1H), 4.52–4.45 (m, 1H), 3.91 (dd,
H,H =7.8, 4.9 Hz, 1H), 3.71 (ddd, JH,H =11.6, 6.1, 4.7 Hz, 1H), 3.56
(ddd, JH,H =12.0, 6.1, 5.8 Hz, 1H), 3.05–2.90 (m, 1H), 2.20 (s, 3H),
2.00–1.86 ppm (m, 1H); 13C NMR (101 MHz, [D6]DMSO): d=168.7,
167.3, 151.3, 150.8, 150.6, 139.5, 135.4, 131.9, 129.5, 128.6, 128.4,
127.4, 126.7, 126.4, 125.7, 119.0, 118.7, 88.4, 84.9, 70.9, 61.9, 38.9,
24.3 ppm; IR (ATR): n˜ =3247, 2919, 1674, 1609, 1583, 1526, 1493,
1456, 1352, 1274, 1176, 1096, 1053, 1028, 986, 943, 907, 880, 855,
782, 753, 728, 693, 677, 647, 616, 597, 569, 548, 504, 462, 416,
390 cmꢀ1; UV (CHCl3/CH3OH 1:1, v/v): lmax =240.4806, 286.5065 nm;
HRMS (ESI+): m/z calcd: 488.1808 [M]+; found: 489.1877 [M+H]+.
N6-Benzoyl-8-(N-acetyl)-4-methoxyphenyl-3’,5’-O-bis(tert-butyldi-
methylsilyl)-2’-deoxyadenosine (14c): GP-III was conducted with
13c (1.01 g, 1.17 mmol), which afforded the desired product as
a yellow foam (1.03 g, 1.29 mmol, 81%). M.p. 958C; [a]25859 nm =34.4
N6-Benzoyl-8-(N-acetyl)-3,5-dimethylphenyl-2’-deoxyadenosine
(15b): GP-IV was conducted with 14b (0.69 g, 0.93 mmol), which
afforded the desired product as a yellow foam (0.37 g, 0.71 mmol,
76%). M.p. 144.48C; [a]52809 nm =220.6 (c=0.5, CHCl3/CH3OH 1:1, v/v);
1H NMR (400 MHz, [D6]DMSO, 323 K): d=10.48 (s, 1H), 8.63 (s, 1H),
1
(c=0.5, CHCl3); H NMR (400 MHz, CDCl3): d=8.63 (s, 1H), 7.54 (d,
J
H,H =6.7 Hz, 2H), 7.45 (t, JH,H =7.4 Hz, 1H), 7.36 (t, JH,H =7.6 Hz, 2H),
7.25 (d, JH,H =8.8 Hz, 2H), 6.94 (d, JH,H =8.4 Hz, 2H), 6.33 (dd, JH,H
7.0, 6.9 Hz, 1H), 4.55–4.46 (m, 1H), 3.92 (dd, JH,H =7.7, 4.7 Hz, 1H),
=
ChemBioChem 2012, 13, 700 – 712
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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709

C. Meier et al.
7.56 (d, JH,H =7.6 Hz, 2H), 7.45 (t, JH,H =7.4 Hz, 1H), 7.36 (t, JH,H
7.7 Hz, 2H), 6.94 (s, 1H), 6.91 (s, 2H), 6.29 (t, JH,H =7.0 Hz, 1H), 5.18
(d, JH,H =4.4 Hz, 1H), 4.99–4.89 (m, 1H), 4.53–4.44 (m, 1H), 3.92 (dd,
=
sodium sulfate and filtered, and the solvent was removed in vacuo.
The residue was purified by flash chromatography on ALOX
(act. III) with methanol in dichloromethane (0!2%) to afford the
desired product.
J
H,H =7.8, 4.8 Hz, 1H), 3.72 (ddd, JH,H =11.7, 4.7, 4.7 Hz, 1H), 3.57
(ddd, JH,H =11.9, 7.0, 5.1 Hz, 1H), 3.04–2.94 (m, 1H), 2.21 (s, 6H),
2.20), 1.99–1.84 ppm (m, 1H); 13C NMR (101 MHz, [D6]DMSO): d=
172.2, 168.7, 150.8, 150.1, 149.4, 140.3, 138.7, 131.6, 131.5, 128.9,
128.5, 128.4, 128.2, 127.7, 119.0, 118.7, 88.4, 84.9, 70.9, 61.9, 38.9,
24.3, 20.6 ppm; IR (ATR): n˜ =3302, 3248, 3101, 2919, 2864, 2363,
2339, 1681, 1609, 1583, 1526, 1498, 1463, 1447, 1350, 1302, 1283,
1263, 1178, 1099, 1082, 1058, 1037, 986, 961, 936, 904, 852, 825,
797, 777, 739, 705, 684, 651, 631, 612, 575, 537, 515, 491, 479, 467,
N6-Benzoyl-8-(N-acetyl)-phenyl-5’-O-dimethoxytrityl-2’-deoxy-
adenosine (16a): GP-V was conducted with 15a (0.28 g,
0.58 mmol), which afforded the desired product as a yellow foam
(0.32 g, 0.41 mmol, 71%). M.p. 1288C; [a]25859 nm =104.4 (c=0.5,
CHCl3/CH3OH 1:1, v/v); H NMR (400 MHz, CDCl3): d=8.60 (s, 1H),
7.56–7.51 (m, 2H), 7.31–7.25 (m, 8H), 7.22–7.18 (m, 3H), 7.18–7.11
1
(m, 5H), 7.84–7.78 (m, 5H), 6.38 (dd, JH,H =7.0, 5.8 Hz, 1H), 4.71 (d,
450, 432, 415, 404, 390 cmꢀ1; UV (CHCl3/CH3OH 1:1, v/v): lmax
=
J
H,H =3.7 Hz, 1H), 4.22–4.15 (m, 1H), 3.78 (s, 6H), 3.57 (dd, JH,H =7.8,
229.9445, 284.0429 nm; HRMS (ESI+): m/z calcd: 516.2121 [M]+;
found: 517.2193 [M+H]+.
4.9 Hz, 1H), 3.12–3.04 (m, 3H), 2.57 (s, 3H), 1.46–1.41 ppm (m, 1H);
13C NMR: (100 MHz, CDCl3): d=159.2, 159.1, 145.9, 151.6, 136.6,
131.6, 131.4, 130.8, 130.6, 130.6, 130.5, 129.8, 129.6, 129.5, 128.8,
128.7, 128.6, 128.3, 113.6, 113.5, 86.8, 85.6, 75.1, 54.8, 43.5, 36.1,
24.7 ppm; IR (ATR): n˜ =3277, 2933, 2836, 1682, 1608, 1582, 1508,
1459, 1400, 1352, 1281, 1247, 1175, 1063, 1030, 951, 828, 790, 755,
726, 696, 644, 581, 549, 504 cmꢀ1; HRMS (ESI+): m/z calcd:
790.3115 [M]+; found: 791.3154 [M+H]+.
N6-Benzoyl-8-(N-acetyl)-4-methoxyphenyl-2’-deoxyadenosine
(15c): GP-IV was conducted with 14c (1.02 g, 1.37 mmol), which af-
forded the desired product as a yellow foam (0.55 g, 1.06 mmol,
78%). M.p. 1518C; [a]52809 nm =227.2 (c=0.5, CHCl3/CH3OH 1:1, v/v);
1H NMR (400 MHz, [D6]DMSO, 323 K): d=10.49 (s, 1H), 8.63 (s, 1H),
7.54 (d, JH,H =6.7 Hz, 2H), 7.45 (t, JH,H =7.4 Hz), 7.36 (t, JH,H =7.6 Hz,
2H), 7.25 (d, JH,H =8.8 Hz, 2H), 6.94 (d, JH,H =8.4 Hz, 2H), 6.33 (dd,
N6-Benzoyl-8-(N-acetyl)-3,5-dimethylphenyl-5’-O-dimethoxytrityl-
2’-deoxyadenosine (16b): GP-V was conducted with 15b (0.66 g,
1.29 mmol), which afforded the desired product as a yellow foam
(0.67 g, 0.83 mmol, 64%). M.p. 1378C; [a]25859 nm =144.6 (c=0.5,
J
H,H =7.0, 6.9 Hz, 1H), 5.20 (d, JH,H =4.4 Hz, 1H), 4.95–4.89 (m, 1H),
4.55–4.46 (m, 1H), 3.92 (dd, JH,H =7.7, 4.7 Hz, 1H), 3.73 (s, 3H),
3.74–3.69 (m, 1H), 3.57 (ddd, JH,H =11.7, 5.9, 4.8 Hz, 1H), 3.06–2.93
(m, 1H), 2.20 (s, 3H), 2.02–1.91 ppm (m, 1H); 13C NMR (101 MHz,
[D6]DMSO): d=172.1, 168.6, 151.4, 150.7, 149.3, 147.5, 139.4, 134.5,
133.8, 133.5, 133.0, 131.3, 128., 128.1, 127.8, 121.7, 114.6, 88.3, 84.8,
70.8, 61.8, 55.2, 37.1, 24.1 ppm; IR (ATR): n˜ =3247, 2931, 2837,
1670, 1607, 1584, 1530, 1507, 1462, 1441, 1355, 1283, 1243, 1172,
1136, 1096, 1053, 1027, 987, 943, 908, 879, 863, 831, 797, 775, 719,
661, 608, 581, 548, 527, 491, 455, 432, 417, 390, 381 cmꢀ1; UV
(CHCl3/CH3OH 1:1, v/v): lmax =248.7985, 307.5786 nm; HRMS (ESI+):
m/z calcd: 518.1914 [M]+; found: 519.1981 [M+H]+.
1
CHCl3/CH3OH 1:1, v/v); H NMR: (400 MHz,CDCl3): d=8.60 (s, 1H),
7.29–7.27 (m, 4H), 7.18–7.15 (m, 7H), 7.03 (s, 1H), 6.95 (s, 2H),
6.85–6.81 (m, 7H), 6.30 (dd, JH,H =9.8, 5.3 Hz, 1H), 4.83–4.76 (m,
1H), 4.27–4.23 (m, 1H), 4.02–3.94 (m, 2H), 3.77 (s, 6H), 3.57–3.50
(m, 1H), 2.29–2.23 (m, 1H), 2.18 (s, 6H), 2.01 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=160.4, 159.9, 159.8, 152.3, 151.2, 150.1, 146.7,
141.0, 140.9, 137.1, 133.0, 132.7, 131.4, 131.4, 131.1, 131.0, 130.6,
130.3, 130.1, 129.9, 129.5, 129.3, 128.5, 127.8, 113.9, 113.8, 113.8,
113.7, 88.8, 86.6, 73.4, 55.7, 55.6, 47.1, 38.0, 24.7, 21.2, 21.2 ppm; IR
(ATR): n˜ =2930, 2835, 1693, 1608, 1582, 1507, 1461, 1368, 1349,
1281, 1245, 1174, 1109, 1073, 1051, 1029, 961, 916, 827, 800, 790,
754, 726, 697, 642, 581, 542, 488, 466, 389 cmꢀ1; HRMS (FAB): m/z
calcd: 818.3428 [M]+; found: 819.3479 [M+H]+.
N6-Benzoyl-8-(N-acetyl)-4-biphenyl-2’-deoxyadenosine (15d): GP-
IV was conducted with 14d (1.20 g, 1.51 mmol), which afforded
the desired product as a yellow foam (0.78 g, 1.38 mmol, 91%).
1
M.p. 1478C; [a]25859 nm =183.8 (c=0.5, CHCl3/CH3OH 1:1, v/v); H NMR
N6-Benzoyl-8-(N-acetyl)-4-methoxyphenyl-5’-O-dimethoxytrityl-
2’-deoxyadenosine (16c): GP-V was conducted with 15c (0.54 g,
1.03 mmol), which afforded the desired product as a yellow foam
(0.37 g, 0.45 mmol, 43%). M.p. 1398C; [a]25859 nm =139.4 (c=0.5,
(400 MHz, [D6]DMSO, 323 K): d=10.54 (s, 1H), 8.64 (s, 1H), 7.71 (d,
J
H,H =6.6 Hz, 1H), 7.66 (d, JH,H =7.3 Hz, 2H), 7.61 (d, JH,H =7.3 Hz,
2H), 7.49–7.42 (m, 3H), 7.40–7.34 (m, 5H), 6.34 (dd, JH,H =7.0,
6.9 Hz, 1H), 5.20 (brs, 1H), 4.90 (brs, 1H), 4.51–4.47 (m, 1H), 3.92
(dd, JH,H =7.9, 4.9 Hz, 1H), 3.72 (dd, JH,H =11.7, 4.4 Hz, 1H), 3.59–
3.54 (m, 1H), 3.05–2.96 (m, 1H), 2.20 (s, 3H), 2.01–1.90 ppm (m,
1H); 13C NMR (101 MHz, [D6]DMSO): d=172.2, 168.6, 151.6, 149.4,
149.1, 138.9, 138.6, 131.7, 131.5, 131.4, 128.8, 128.3, 128.2, 127.6,
127.4, 126.5, 126.4, 119.0, 118.7, 88.3, 84.9, 70.8, 61.8, 39.0,
24.1 ppm; IR (ATR): n˜ =3270, 3063, 3031, 2933, 2861, 1678, 1607,
1583, 1518, 1485, 1460, 1448, 1402, 1348, 1319, 1276, 1176, 1098,
1055, 1025, 1007, 988, 944, 839, 788, 763, 719, 694, 665, 640, 596,
555, 503, 466, 398 cmꢀ1; UV (CHCl3/CH3=H 1:1, v/v): lmax =249.9076,
302.0333 nm; HRMS (FAB): m/z calcd: 564.2121 [M]+; found:
565.2196 [M+H]+
1
CHCl3/CH3OH 1:1, v/v); H NMR (400 MHz, CDCl3): d=8.61 (s, 1H),
7.57–7.53 (m, 2H), 7.44–7.38 (m, 1H), 7.32–7.27 (m, 7H), 7.19–7.13
(m, 6H), 6.85–6.81 (m, 6H), 6.40 (dd, JH,H =9.7, 5.3 Hz, 1H), 4.76–
4.71 (m, 1H), 4.23–4.20 (m, 1H), 4.01–3.95 (m, 2H), 3.80 (s, 6H),
3.78 (s, 3H), 2.58 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
165.3, 158.0, 154.7, 152.8, 150.4, 147.9, 145.0, 144.6, 135.8, 135.6,
134.0, 132.8, 129.7, 129.6, 128.4, 128.3, 127.6, 126.5, 126.2, 120.8,
113.8, 113.0, 86.6, 84.7, 74.7, 64.1, 55.2, 55.1, 35.1, 24.7 ppm; IR
(ATR): n˜ =3356, 3057, 2999, 2932, 2835, 2361, 1735, 1670, 1609,
1563, 1462, 1444, 1372, 1301, 1177, 1034, 910, 828, 791, 754, 726,
617, 584 cmꢀ1
;
MS (FAB): m/z calcd: 820.3221 [M]+; found:
821.3263 [M+H]+.
General Procedure V for the 5’-O-dimethoxytritylation of the N6-
(Bz)-8-(N-ac)-arylamine-2’-dA adducts to afford compounds 16a–
d: The appropriate compound 15 was dissolved in anhydrous pyri-
dine (20 mL) under nitrogen and 4,4’-dimethoxytrityl chloride
(1 equiv) and silver nitrate (2 equiv) were added. The mixture was
stirred at room temperature until the reaction was complete
(ꢁ3 h). It was then diluted with dichloromethane and washed with
brine, and the aqueous layer was extracted three times with di-
chloromethane. The combined organic layers were dried over
N6-Benzoyl-8-(N-acetyl)-4-biphenyl-5’-O-dimethoxytrityl-2’-de-
oxyadenosine (16d): GP-V was conducted with 15d (0.77 g,
1.36 mmol), which afforded the desired product as a yellow foam
(0.74 g, 0.85 mmol, 63%). M.p. 1608C; [a]25859 nm =145.8 (c=0.5,
1
CHCl3/CH3OH 1:1, v/v); H NMR (400 MHz, CDCl3): d=8.52 (s, 1H),
7.52–7.43 (m, 8H), 7.35–7.30 (m, 4H), 7.28–7.23 (m, 4H), 7.19–7.17
(m, 3H), 7.09–7.05 (m, 4H), 6.75–6.72 (m, 4H), 6.32 (dd, JH,H =9.7,
5.3 Hz, 1H), 4.66–4.61 (m, 1H), 4.13–4.10 (m, 1H), 3.92–3.83 (m,
710
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 700 – 712

DNA Damage by Aromatic Amines
2H), 3.70 (s, 6H), 2.49 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=
163.1, 152.4, 147.1, 142.1, 140.9, 139.8, 139.6, 133.9, 131.5, 129.9,
129.3, 129.2, 129.0, 128.2, 128.1, 128.0, 128.0, 127.3, 127.2, 113.3,
87.5, 82.8, 67.1, 61.1, 55.4, 31.1 ppm; IR (ATR): n˜ =3033, 2966, 2932,
2837, 2251, 1682, 1608, 1583, 1508, 1487, 1461, 1447, 1409, 1353,
1319, 1281, 1247, 1176, 1157, 1127, 1065, 1029, 1008, 976, 901, 828,
790, 764, 724, 698, 666, 640, 581, 562, 523 cmꢀ1; HRMS (FAB): m/z
calcd: 866.3428 [M]+; found: 867.3457 [M+H]+.
N6-Benzoyl-[8-(N-acetyl)-4-methoxyphenylamino]-5’-O-dimeth-
oxytrityl-2’-deoxyadenosine-3’-O-(b-cyanoethyl-N,N’-diisopropyl-
phosphoramidite (17c): GP-VI was conducted with 16c (0.35 g,
0.43 mmol), which afforded the desired product as a colourless
solid (0.28 g, 0.28 mmol, 63%). M.p. 968C; [a]25809 nm =84.6 (c=0.5,
CHCl3); 1H NMR (400 MHz, C6D6): d=8.29 (s, 1H), 7.91–7.89 (m,
2H), 7.67–7.51 (m, 6H), 7.48–7.31 (m, 10H), 7.13–6.91 (m, 12H),
6.78–6.75 (m, 12H), 6.56–6.48 (m, 1H), 5.20–4.91 (m, 1H), 4.00–3.75
(m, 2H), 3.50–3.40 (m, 2H), 3.33 (s, 6H), 3.30–3.20 (m, 2H), 1.90 (s,
6H), 1.80–1.63 (m, 2H), 1.19–1.15 (m, 1H), 1.12 (s, 3H), 1.10 (s, 3H),
1.11 (s, 3H), 0.99 ppm (s, 3H); 13C NMR (100 MHz, C6D6): d=159.2,
159.1, 155.6, 153.3, 146.0, 139.5, 136.7, 136.6, 131.4, 130.8, 130.7,
129.4, 129.3, 128.5, 127.0, 113.6, 113.5, 87.9, 86.1, 77.4 59.1, 54.8,
43.6, 36.3, 24.7, 24.6, 24.5, 24.5, 21.1 ppm; 31P NMR (162 MHz,
[D6]benzene): d=162.64, 162.45, 161.81, 161.74 ppm; IR (ATR): n˜ =
3033, 2964, 2932, 2836, 2545, 2389, 2250, 2220, 2181, 2163, 2143,
2111, 2049, 2008, 1977, 1894, 1680, 1607, 1582, 1508, 1461, 1446,
1396, 1364, 1286, 1246, 1201, 1176, 1156, 1126, 1029, 977, 900, 828,
791, 772, 754, 724, 701, 681, 664, 641, 581, 548, 523, 461, 428, 410,
392 ppm; MS (FAB): m/z calcd: 1020.4 [M]+; found: 1022.3
[M+2H]+.
General Procedure VI for the 3’-O-phosphitylation of the N6-
(benzoyl)-8-(N-acetyl)-arylamine-5’-O-DMTr-2’-dA adducts to af-
ford compounds 17a–d: The appropriate compound 16 was dis-
solved in anhydrous CH2Cl2 (5 mL) and acetonitrile (5 mL) under
nitrogen, and DCI (1.0 equiv) was added. The reaction mixture was
treated with bis-N,N’-diisopropylamino-(2-O-cyanoethyl)phosphate
(1.5 equiv). The mixture was stirred at room temperature until the
reaction was complete (ꢁ2 h). It was then diluted with dichloro-
methane and washed with water. The aqueous layer was extracted
three times with CH2Cl2. The combined organic layers were dried
over sodium sulfate and filtered, and the solvent was removed in
vacuo. The residue was purified by flash chromatography on ALOX
(act. III) with methanol in CH2Cl2 (0!1%). The product was redis-
solved in benzene and gave the desired product as a colourless
solid after freeze-drying.
N6-Benzoyl-[8-(N-acetyl)-4-biphenylamino]-5’-O-dimethoxytrityl-
2’-deoxyadenosine-3’-O-(b-cyanoethyl-N,N’-diisopropylphosphor-
amidite (17d): GP-VI was conducted with 16d (0.72 g, 0.83 mmol),
which afforded the desired product as a colourless solid (0.51 g,
0.48 mmol, 58%). M.p. 60.48C; [a]25809 nm =166 (c=0.5, CHCl3);
1H NMR (400 MHz, C6D6): d=8.05 (s, 1H), 7.71–7.63 (m, 2H), 7.54–
7.46 (m, 4H), 7.33–7.20 (m, 4H), 7.20–6.99 (m, 12H), 6.95–6.88 (m,
3H), 6.72–6.60 (m, 6H), 4.78–4.70 (m, 1H), 3.86–3.75 (m, 2H), 3.48–
3.39 (m, 1H), 3.30 (s, 3H), 3.28 (s, 3H), 3.15–3.01 (m, 2H), 2.96–2.88
(m, 2H), 2.38 (s, 3H), 1.53–1.46 (m, 2H), 1.09–0.99 ppm (m, 12H);
13C NMR (100 MHz, C6D6): d=159.2, 149.7, 141.1, 136.0, 135.9,
134.9, 132.6, 131.9, 130.8, 130.6, 129.0, 128.8, 128.7, 128.6, 127.9,
127.1, 127.0, 119.7, 117.6, 113.6, 87.0, 85.8, 59.3, 58.0, 45.4,
20.6 ppm; 31P NMR (162 MHz, C6D6): d=162.71, 162.67, 161.91,
161.84 ppm; IR (ATR): n˜ =3033, 2966, 2932, 2837, 2251, 1682, 1608,
1583, 1508, 1487, 1461, 1447, 1409, 1353, 1319, 1281, 1247, 1176,
1157, 1127, 1065, 1029, 1008, 976, 901, 828, 790, 764, 724, 698,
666, 640, 581, 562, 523 cmꢀ1; MS (FAB): m/z calcd: 1066.3 [M]+;
found: 1068.2 [M+2H]+.
N6-Benzoyl-[8-(N-acetyl)-phenylamino]-5’-O-dimethoxytrityl-2’-
deoxyadenosine-3’-O-(b-cyanoethyl-N,N’-diisopropylphosphora-
midite (17a): GP-VI was conducted with 16a (0.31 g, 0.40 mmol),
which afforded the desired product as a colourless solid (0.30 g,
1
0.30 mmol, 76%). M.p. 958C; H NMR (400 MHz, C6D6): d=8.33 (s,
1H), 7.72–7.65 (m, 5H), 7.55–7.47 (m, 5H), 7.15–7.10 (m, 4H), 6.77–
6.62 (m, 9H), 6.63–6.55 (m, 1H), 5.18–5.00 (m, 1H), 4.75–4.62 (m,
1H), 3.88–3.76 (m, 2H), 3.56–3.42 (m, 2H), 3.33 (s, 6H), 3.27–3.17
(m, 2H), 2.39 (s, 3H), 1.82–1.67 (m, 2H), 1.09–0.98 ppm (m, 12H);
13C NMR (101 MHz, C6D6): d=163.8, 159.2, 151.7, 151.6, 149.9,
146.7, 145.8, 136.5, 136.4, 134.5, 133.9, 131.9, 131.6, 131.5, 130.7,
130.5, 129.7, 129.5, 128.6, 128.5, 127.4, 127.1, 122.6, 113.3, 86.9,
85.6, 74.9, 64.8, 59.0, 54.8, 43.6, 36.1, 31.1, 24.7, 24.6, 20.0 ppm;
31P NMR (162 MHz, C6D6): d=162.78, 162.69, 161.88, 161.83 ppm;
IR (ATR): n˜ =2965, 2931, 2836, 1678, 1635, 1604, 1578, 1508, 1460,
1446, 1410, 1396, 1366, 1327, 1293, 1248, 1200, 1177, 1155, 1125,
1057, 1030, 1002, 977, 900, 879, 828, 791, 754, 725, 697, 643, 582,
551, 521 cmꢀ1; MS (ESI+): m/z calcd: 990.4 [M]+; found: 990.5
[M+H]+.
Oligonucleotide synthesis: Oligonucleotides were synthesised on
a 1 mmol scale with use of Pac-protected dA, iPrPac-protected dG,
ac-protected dC and T phosphoramidites on a 394 DNA synthesiser
(Applied Biosystems) and of phosphoramidites and solid supports
purchased from ChemGenes. The manufacturer’s standard synthe-
sis protocol was used, except that on incorporation of the modi-
fied phosphoramidites the coupling was repeated three times,
each for 500 s. The oligonucleotides were purified by HPLC with
triethylammonium acetate buffer (pH 6.9, solvent 1) and acetoni-
trile (solvent 2) on a C-18 reversed-phase column with UV detec-
tion. The solvent gradient was as follows: initially 99% solvent 1,
then a 50 min linear gradient to 23% solvent 2; 10 min with 100%
solvent 2; 10 min with 100% solvent 1.
N6-Benzoyl-[8-(N-acetyl)-3,5-dimethylphenylamino]-5’-O-dimeth-
oxytrityl-2’-deoxyadenosine-3’-O-(b-cyanoethyl-N,N’-diisopropyl-
phosphoramidite (17b): GP-VI was conducted with 16b (0.66 g,
0.81 mmol), which afforded the desired product as a colourless
solid (0.29 g, 0.29 mmol, 36%). M.p. 1038C; [a]25809 nm =304.0 (c=0.5,
CHCl3); 1H NMR: (400 MHz, C6D6): d=8.26 (s, 1H), 7.70–7.68 (m,
2H), 7.53–7.51 (m, 4H), 7.13–7.09 (m, 3H), 7.05–7.02 (m, 2H), 6.94–
6.80 (m, 3H), 6.72–6.62 (m, 6H), 6.56–6.48 (m, 1H), 5.20–4.91 (m,
3H), 4.69 (m, 1H), 4.00–3.75 (m, 2H), 3.50–3.40 (m, 2H), 3.33 (s,
6H), 3.30–3.20 (m, 2H), 1.90 (s, 6H), 1.80–1.63 (m, 2H), 1.19–1.15
(m, 1H), 1.12 (s, 3H), 1.10 (s, 3H), 1.11 (s, 3H), 0.99 ppm (s, 3H);
13C NMR: (100 MHz, C6D6): d=159.2, 159.1, 155.6, 153.3, 146.0,
139.5, 136.7, 136.6, 131.4, 130.8, 130.7, 129.4, 129.3, 128.5, 127.0,
113.6, 113.5, 87.9, 86.1, 77.4, 59.1, 54.8, 43.6, 36.3, 24.7, 24.6, 24.5,
24.5, 21.1 ppm; 31P NMR (162 MHz, C6D6): d=162.60, 162.58,
161.77, 161.72 ppm; IR (ATR): n˜ =2965, 2929, 2872, 2836, 1697,
1634, 1607, 1508, 1462, 1445, 1396, 1365, 1327, 1296, 1248, 1200,
1177, 1156, 1116, 1073, 1031, 976, 899, 879, 828, 800, 770, 754, 725,
703, 681, 641, 583, 521, 463, 393 cmꢀ1; MS (FAB): m/z calcd: 1018.4
[M]+; found: 1019.3 [M+H]+.
HRMS data for the oligonucleotides are given in Table 5.
Thermal melting studies: Equal amounts of the two complemen-
tary strands (2 nmol) were dissolved in buffer [1 mL, phosphate
buffer (10 mm), NaCl (140 mm), EDTA (1 mm), pH 6.6]. The UV ab-
sorption at 260 nm was monitored as a function of temperature.
The temperature was increased at a rate of 0.58Cminꢀ1 over the
range 5–808C.
ChemBioChem 2012, 13, 700 – 712
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
711

C. Meier et al.
of [g32P]-ATP by standard techniques. Annealing of the primer to
the template strand was conducted in Tris·HCl (pH 7.5, 20 mm) and
NaCl (50 mm) from 958C to 258C over at least 1 h. Mixtures were
incubated for 30 min at different temperatures (688C for Pfu DNA
polymerase, 378C for human DNA polymerase ß and for Dpo4 DNA
polymerase) and the reactions were stopped by the addition of
stop solution [45 mL, formamide (80%, v/v), EDTA (20 mm), bromo-
phenol blue (0.025%, w/v), xylene cyanol (0.025%, w/v)]. The mix-
tures were separated by denaturating PAGE (12%) followed by
visualisation through phosphorimaging.
Table 5. HRMS data for oligonucleotides 18a to 20j.
Oligonucleotide
m/z calcd
m/z found
18a (MALDI-TOF)
18b (MALDI-TOF)
18c (MALDI-TOF)
18d (MALDI-TOF)
18e (MALDI-TOF)
18 f (MALDI-TOF)
18g (MALDI-TOF)
18h (MALDI-TOF)
18i (MALDI-TOF)
19a (HPLC-ESI)
19b (HPLC-ESI)
19c (HPLC-ESI)
19d (HPLC-ESI)
19e (HPLC-ESI)
19 f (HPLC-ESI)
19g (HPLC-ESI)
19h (HPLC-ESI)
19i (HPLC-ESI)
19j (HPLC-ESI)
19k (HPLC-ESI)
19l (HPLC-ESI)
19m (HPLC-ESI)
20a (HPLC-ESI)
20b (HPLC-ESI)
20d (HPLC-ESI)
20 f (HPLC-ESI)
20h (HPLC-ESI)
20j (HPLC-ESI)
3566.4
3657.4
3700.4
3687.5
3730.4
3686.5
3729.5
3733.5
3776.4
3644.5
3735.5
3778.5
3765.5
3808.5
3811.5
3854.5
3735.5
3778.5
3765.5
3808.5
3811.5
3854.5
6095.0
6186.0
6215.1
6216.1
6262.1
6274.1
3566.2
3656.0
3699.1
3688.1
3729.1
3685.0
3729.0
3735.0
3777.1
1214.8 (ꢀ3)
1245.2 (ꢀ3)
1259.5 (ꢀ3)
1255.2 (ꢀ3)
1269.5 (ꢀ3)
1270.6 (ꢀ3)
1284.8 (ꢀ3)
1244.2 (ꢀ3)
1257.5 (ꢀ3)
1255.9 (ꢀ3)
1268.2 (ꢀ3)
1270.6 (ꢀ3)
1284.8 (ꢀ3)
2030.3 (ꢀ3)
2061.0 (ꢀ3)
1552.7 (ꢀ4)
2070.7 (ꢀ3)
2086.0 (ꢀ3)
2090.0 (ꢀ3)
Acknowledgements
C.M. and A.M. are grateful for financial support from the Deut-
sche Forschungsgemeinschaft (ME 1161/9–1 and MA 2288/8–1)
and the University of Hamburg.
Keywords: aromatic amines · chemical carcinogenesis · DNA
adducts · DNA damage · electrophilic amination
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4697.
Primer-extension reactions: The recombinant enzymes were puri-
fied as described in the literature. Pfu DNA polymerase: see
ref. [26]. Human DNA polymerase b: see ref. [27]. Dpo4 DNA poly-
merase: adapted from ref. [28]. The reaction mixtures (10 mL) con-
tained 32P-labelled primer F15 (5’-CGCGTC GTAGAT GCT-3’,
100 nm), the different templates (150 nm) and dNTPs (100 mm) in
1ꢂ reaction buffer [for Pfu DNA polymerase: Tris·HCl (pH 8.8,
20 mm), (NH4)2SO4 (10 mm), KCl (10 mm), MgSO4 (2 mm), Triton X-
100 (0.1%, v/v), BSA (0.2%, w/v); for human DNA polymerase b:
Tris·HCl (pH 8.0, 50 mm), MgCl2 (10 mm), DTT (2 mm), NaCl (20 mm),
KCl (20 mm), glycerol (1%, v/v); for Dpo4 DNA polymerase: Tris·HCl
(pH 7.8, 50 mm), DTT (5 mm), NaCl (50 mm), MgCl2 (5 mm), glycerol
(5%, v/v)]. The final enzyme concentrations in the reaction mix-
tures were 10 nm for Pfu DNA polymerase, 200 nm for human DNA
polymerase b and 50 nm for Dpo4 DNA polymerase. The template
sequences used are depicted in Figure 6. F15 was labelled by use
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Received: September 6, 2011
Published online on February 29, 2012
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