Synthesis of site-specific damaged DNA strands by 8-(acetylarylamino)- 2′-deoxyguanosine adducts and effects on various DNA polymerases

Article abstract of DOI:10.1002/ejoc.201200984

Beside the predominately found 8-(arylamino)-2′-dG, 8-(acetylarylamino) damages within DNA-strands may also play an important role in the induction of chemical carcinogenesis. A synthesis pathway leading to these 8-(acetylarylamino)-dG adducts using different aromatic amines has been optimized. The 8-modified dGs were converted into the corresponding phosphoramidites and site-specifically incorporated into different oligonucleotides leading to DNA strands. Lesion-bearing hybrids of these damaged DNA-strands with complementary oligonucleotides were used to study their melting properties and their circular dichroism spectra. It was shown that no EcoRI restriction took place with the damage inside the cleavage site. Finally, three different DNA polymerases were used for primer extension studies. C8-NAc-Arylamine adducts of 2′-deoxyguanosine with various aromatic amines were synthesized by using cross-coupling reactions and converted into 3′-phosphoramidites. Site-specific damaged NarI-, EcoRI- and 20mer-oligonucleotides were prepared by automated DNA-synthesis. Biophysical properties, restriction endonuclease studies and DNA-polymerase assays were performed. Copyright

Full text of DOI:10.1002/ejoc.201200984

FULL PAPER
DOI: 10.1002/ejoc.201200984
Synthesis of Site-Specific Damaged DNA Strands by 8-(Acetylarylamino)-2Ј-
deoxyguanosine Adducts and Effects on Various DNA Polymerases
Sarah Krüger[a] and Chris Meier*[a]
Keywords: DNA / DNA damage / Carcinogenesis / Cross-coupling / Amines / Electrophilic addition
Beside the predominately found 8-(arylamino)-2Ј-dG, 8-
(acetylarylamino) damages within DNA-strands may also
play an important role in the induction of chemical carcino-
genesis. A synthesis pathway leading to these 8-(acetylaryl-
amino)-dG adducts using different aromatic amines has been
optimized. The 8-modified dGs were converted into the cor-
responding phosphoramidites and site-specifically incorpo-
rated into different oligonucleotides leading to DNA strands.
Lesion-bearing hybrids of these damaged DNA-strands with
complementary oligonucleotides were used to study their
melting properties and their circular dichroism spectra. It was
shown that no EcoRI restriction took place with the damage
inside the cleavage site. Finally, three different DNA poly-
merases were used for primer extension studies.
Introduction
Polycyclic aromatic amines, such as 2-aminofluorene, 2-
(acetylamino)fluorene (2-AAF), 4-aminobiphenyl and 4-
(acetylamino)biphenyl are well-known chemical carcino-
gens.[1–6] These chemicals are metabolically activated in cells
to give highly-reactive arylnitrenium-ions that react with
cellular DNA to form covalent adducts predominantly at
the 8-position of 2Ј-deoxyguanosine (dG) residues by means
of an electrophilic amination reaction. The aromatic amines
form two major DNA adducts, the 8-(arylamino)-2Ј-dG (1)
and the 8-(acetylarylamino)-2Ј-dG (2) (Figure 1). In con-
trast to the polycyclic aromatic amines, their monocyclic
counterparts are less toxic and thus they were classified as
borderline carcinogens. However, aniline derivatives are
ubiquitously present in the environment originating from
many sources.[7–10] Interestingly, monocyclic aromatic
amines are metabolized through the same enzymatic path-
ways and give the same type of DNA damage relative to
the polycyclic aromatic amines, particularly in the case of
donor-substituted derivatives. Recently, owing to the
ubiquitous presence of anilines, the potential human carci-
nogenicity of these compounds has regained interest.[11] Al-
though these two adducts differ only by an N-acetyl group,
they have very different physicochemical properties and bio-
logical effects.
Figure 1. Chemical structure of 8-(arylamino)-2Ј-dG (1) and 8-
(acetylarylamino)-2Ј-dG (2).
The acetylated form causes a much more pronounced lo-
cal distortion within the DNA double-helix than the non-
acetylated form.[5,12] This was correlated with a conforma-
tional difference of these two adducts. Whereas 8-NH-2Ј-
dG adducts (1) preferred the anti-conformation of the gly-
cosidic bond placing the aromatic moiety in the major
groove of the double helix and thus not influencing the hy-
drogen-bridge interaction within the DNA double helix, 8-
(acetylarylamino)-2Ј-dG adducts (2) preferred the syn-con-
formation that places the aromatic system within the double
helix and displaces the heterocyclic nucleobase from the he-
lix into the major groove resulting in loss of hydrogen-bond
stabilization.[13,14] It has been reported that the 2-AAF ad-
duct is a more potent blocker of replication and transcrip-
tion; however, it is also a better substrate for DNA-repair
enzymes.[15,16]
[a] Organic Chemistry, Department of Chemistry, Faculty of
Sciences, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg
Fax: +49-40-42838-5592
Zhou and Romano reported on the synthesis of 8-(aryl-
amino)-2Ј-deoxyguanosine phosphoramidite reagents of 2-
aminofluorene and its N-acetyl counterpart for the site-spe-
cific synthesis of oligonucleotide strands containing these
adducts. As a protecting group for the exocyclic amino
E-mail: chris.meier@chemie.uni-hamburg.de
Homepage: http://www.chemie.uni-hamburg.de/oc/meier/
index.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200984.
1158
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Synthesis and Effects of Specific-Site Damaged DNA Strands
function of dG, the fluorenylmethyloxycarbonyl (Fmoc) acetic acid led to an efficient cleavage of the TBDMS
group was used.[17,18]
groups without loss of the N-acetyl group and gave com-
A strategy to prepare the 8-(acetylarylamino)-dG ad- pounds 8a–f in very good yields.
ducts was introduced by Gillet and Schärer in 2005.[19,20]
At this step, the syn- or anti-preference of the glycosidic
They reported the successful conversion of N-acetylated ad- bond for the acetylated and 8-(arylamino)-dG adducts was
ducts into corresponding phosphoramidites (12 steps over- studied by NOESY-NMR spectroscopy. As described ear-
all) and their site-specific incorporation into DNA-oligonu- lier for the corresponding 8-(acetylarylamino)deoxyadenos-
cleotides. It was proven that the N-acetyl group was not ine adducts (syn),[23] the conformational preferences for the
cleaved during the final deprotection.
N-acetylated dG adducts was also found to be the syn-con-
In 2006, we published the synthesis and the site-specific formation. In contrast, both the 8-NH-arylamine-2Ј-deoxy-
incorporation of 8-(arylamino)-modified 2Ј-dG-phos- guanosine and -2Ј-deoxyadenosine adducts showed a pre-
phoramidites into oligonucleotides.[21] Also, we reported on ferred anti-conformation.
the first synthesis of 8-arylamino-2Ј-dA adducts and their
For the oligonucleotide synthesis, the N2-position was se-
successful conversion into the corresponding phosphor- lectively blocked using the formamidine group (mild depro-
amidites as well as their site-specific incorporation into an tection strategy). The formamidine group has been used
oligonucleotide.[22] This work was recently extended to a previously and we have recently used this group successfully
comparative study between 8-(arylamino)- and 8-(acetyl- for 8-NH-arylamine-2Ј-dG oligonucleotides as well.[21,28]
arylamino)-2Ј-deoxyadenosine-bearing oligonucleotides.[23] After N2-protection, the 5Ј-hydroxy function was blocked
Later, we reported a simple route for the non-optimized with a 4,4Ј-dimethoxytrityl group (DMTr) and correspond-
synthesis of the 8-(acetylarylamino)-2Ј-dG adducts and ing phosphoramidites 11a–f were synthesized using pre-
their phosphoramidites.[24] At that time the prepared amid- viously described conditions.[22,29] It is worth mentioning
ites were not used for the synthesis of oligonucleotides.[24] that after conversion of compounds 9 to give DMTr-pro-
Here, we report an efficient synthesis of 8-(acetylaryl- tected nucleosides 10 the purification was also optimized
amino)-2Ј-dG adducts, an improved synthesis of 3Ј-phos- leading to markedly higher chemical yields.[24] For all N-
phoramidites, and their use in automated solid-phase DNA acetylated compounds, line broadening in the 1H NMR
synthesis to give site-specific-modified oligonucleotides of spectra at room temperature was observed owing to the
several mixed sequences containing multiple dGs. In ad- partial double-bond character of the N-acetyl group at the
dition to CD- and Tm measurements, the influence of these 8-position (see also Supporting Information).[22]
adducts on the cleavage of a lesion-bearing DNA duplex by
the endonuclease EcoRI relative to the 8-(arylamino)-dG
adducts was investigated. Moreover, template 20-mer DNA-
oligonucleotides bearing N-8-(acetylarylamino)-dG lesions
were prepared and studied with regard to the primer exten-
sion by three different DNA polymerases.
Site-Specific Synthesis of Oligonucleotides Containing 8-
(N-Ac-aa)-2Ј-dG Adducts of Different Arylamines
To incorporate the 8-(acetylarylamino)-dG lesions into
the oligonucleotides, phosphoramidites 11a–f were dis-
solved in acetonitrile (0.1 m solutions) and the coupling step
of the lesion-bearing-2Ј-dG phosphoramidite building
blocks were repeated three times (every 500 s) with a total
Results and Discussion
Synthesis of 8-(Acetylarylamino)-2Ј-deoxyguanosine
[8-(Ac-aa)-2Ј-dG] Adducts
As summarized in Scheme 1, the hydroxyl groups of 2Ј- coupling efficiency of 70–90%. For the non-modified nu-
dG 3 were protected using tert-butyldimethylsilyl (TBDMS) cleosides commercially available phosphoramidites were
chloride. The O6-position was activated with (1H-benzotri- used.
azol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
Three different site-specifically modified oligonucleotide-
phosphate (BOP) giving intermediate 4.[24,25] The key step sequences were prepared: NarI oligonucleotide [5Ј-d(CTC
of the sequence is the reaction between O6-(1H-benzotri- GGC GCC ATC)-3Ј], a palindromic 12mer-EcoRI oligo-
azol-1-yl)-2Ј-dG 4 and N-acetyl-hydroxamic acids 6a–f that nucleotide [5Ј-d(GTA GAA TTC TAC)-3Ј] as well as a
were prepared by partial reduction of nitrobenzenes 5a–f to 20mer-oligonucleotide [5Ј-d(ACA TGA GCA TCT ACG
give N-arylhydroxylamines followed by N-acetylation with ACG CG)-3Ј] needed for DNA polymerase catalyzed
acetyl chloride.[24,26,27] The target compounds 8-(acetylaryl- primer extension assays.
amino) adducts 7a–f were isolated in 70–95% yield. For the
The final basic deprotection step with concentrated am-
conversion of 7a–f into the corresponding phosphoramid- monia was completed within 2 h at 45 °C. To avoid oxidat-
ites 11a–f, the hydroxy groups were first deprotected. Owing ive side reactions during the deprotection as observed be-
to the introduced N-acetyl group, the removal of the fore in the case of oligonucleotides bearing 8-aminofluo-
TBDMS groups had to be done under mild acid conditions. rene-dG adduct, β-mercaptoethanol was always added to
In contrast to our previous report in which NEt3–3HF and the deprotection solution.[30–32] The obtained oligonucleo-
a different chromatographic purification procedure was tides were purified by reversed-phase HPLC and charac-
used,[24] tetra-n-butylammonium fluoride buffered with terized by ESI-mass spectrometry (neg. mode).
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Scheme 1. Synthesis of the 8-(acetylarylamino)-2Ј-deoxyadenosine adducts and their phosphoramidites.
Table 1. Tm values of NarI-oligonucleotides 12a–i.[a]
Melting Temperature (Tm) and Circular Dichroism Studies
Oligonucleotide
Tm [°C]
Hybrids of the oligonucleotides bearing the 8-(acetylaryl-
amino)-dG adducts as opposed to the 8-(arylamino)-dG
adducts at two different positions with the complementary
strands were formed and the effect on the thermal stability
of these hybrid strands was measured by UV melting-tem-
perature analysis. The data for NarI-oligonucleotides 12a–i
is given in Table 1.
For 8-(arylamino)-damaged 2Ј-dG oligonucleotides 12b,
12d, 12f and 12h a decrease of approximately 7–9 °C was
observed relative to the Tm value of reference NarI-oligonu-
5Ј-d(CTC GGC GCC ATC) –3Ј 12a
58.2
50.3
41.9
49.8
51.5
49.2
44.1
48.8
44.7
5Ј-d[CTC G(phenyl)GC GCC ATC] –3Ј 12b
5Ј-d[CTC G(Ac-phenyl)GC GCC ATC] –3Ј 12c
5Ј-d[CTC G(2-fluorenyl)GC GCC ATC] –3Ј 12d
5Ј-d[CTC G(Ac-2-fluorenyl)GC GCC ATC] –3Ј 12e
5Ј-d[CTC GGC G(phenyl)CC ATC] –3Ј 12f
5Ј-d[CTC GGC G(Ac-phenyl)CC ATC] –3Ј 12g
5Ј-d[CTC GGC G(2-fluorenyl)CC ATC] –3Ј 12h
5Ј-d[CTC GGC G(Ac-2-fluorenyl)CC ATC] –3Ј 12i
[a] Conditions: 1 nmol NarI-oligonucleotide, 1 nmol complemen-
tary oligonucleotide strand, 10 mm phosphate-buffer pH 7.2,
cleotide 12a (Tm = 58.2 °C). However, for the correspond- 140 mm NaCl, 1.0 mm ethylenediaminetetraacetic (EDTA).
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Synthesis and Effects of Specific-Site Damaged DNA Strands
ing 8-(acetylarylamino)-lesions, a decrease in thermal sta-
A significant decrease in the thermal stabilities of dG-
bility of approximately 13–16 °C relative to unmodified lesion-bearing oligonucleotides 13b–s (Tm values decreased
NarI-oligonucleotide 12a was determined. The decrease of by 8–34 °C) relative to reference oligonucleotides 13a (Tm
thermal stability was nearly independent on the position of = 42.1 °C) was observed. Regarding the 8-(arylamino)-dG
the lesion within the oligonucleotide. Surprisingly, for the lesions, there was a marked thermal destabilization of
oligonucleotide bearing 8-[acetyl(2-fluorenyl)amino]-2Ј-dG Ͼ 28 °C if 8-(acetylarylamino)-lesions 13c, 13e, 13g, 13i,
lesion 12e a decrease of only 7 °C in thermal stability was 13k, and 13m were present within the restriction site relative
observed. Maybe the distortion within the double helix is to non-modified reference oligonucleotide 13a. However, no
partly compensated for by the large, polynuclear aromatic significant difference between the oligonucleotide damaged
system of the fluorenyl residue.
by monocyclic aromatic amines and those damaged by
For the palindromic EcoRI-oligonucleotides, the effect polycyclic aromatic amines was detected. On the other
on the Tm values was expected to be more pronounced be- hand, a smaller decrease in thermal stability was observed
cause, after duplex formation, one lesion is present in each when the lesions were introduced at the 5Ј end of the oligo-
strand. Again, the lesions were introduced at two different nucleotide. Here, acetylated monocyclic DNA-damaged 13o
positions within the oligonucleotide. The first lesion was at led to a decrease of 18 °C (Tm = 23.8 °C), whereas the DNA
the site of cleavage of the restriction endonuclease (4th base damage caused by the polycyclic aromatic residue in 13s
from the 5Ј end) and the second was at the 5Ј terminus, has a smaller influence (Tm = 26.2 °C). The Tm values of
thus outside the restriction region. The Tm values are sum- the NH adducts showed the opposite effect. The highest
marized in Table 2.
influence in thermal stability was observed with the strong
carcinogen biphenylamine (in 13r), whereas the borderline
carcinogen aniline (in 13n) showed a less significant de-
crease in thermal stability. The hybridization data for
20mer-oligonucleotides 14a–m is summarized in Table 3.
The double strand of unmodified reference 20mer-oligo-
nucleotide 14a showed a thermal stability of 68.5 °C. A de-
crease in the thermal stabilities of dG-adduct-bearing oligo-
nucleotides 14b–m (Tm values decreased by 2–13 °C) rela-
tive to the reference oligonucleotide was observed. For 8-
(arylamino)- or 8-(acetylarylamino)-dG oligonucleotides
14h–m, modified with strong carcinogens, only a small de-
crease of the Tm value was observed relative to reference
oligonucleotide 14a. In contrast, the oligonucleotides dam-
aged by acetylated borderline carcinogens 14c and 14e
showed higher destabilization of the duplex stability, which
suggests a larger local distortion caused by the N-acetylated
dG-lesions.
Table 2. Tm values of EcoRI-oligonucleotides 13a–s.[a]
Oligonucleotide
Tm [°C]
5Ј-d(GTA GAA TTC TAC) –3Ј 13a
42.1
5Ј-d[GTA G(phenyl)AA TTC TAC] –3Ј 13b
5Ј-d[GTA G(Ac-phenyl)AA TTC TAC] –3Ј 13c
5Ј-d[GTA G(4-methylphenyl)AA TTC TAC] –3Ј 13d
5Ј-d[GTA G(Ac-4-methylphenyl)AA TTC TAC] –3Ј 13e
5Ј-d[GTA G(4-methoxyphenyl)AA TTC TAC] –3Ј 13f
5Ј-d[GTA G(Ac-4-methoxyphenyl)AA TTC TAC] –3Ј 13g 11.0
5Ј-d[GTA G(3,5-dimethylphenyl)AA TTC TAC] –3Ј 13h
29.5[28]
11.9
23.2[33]
13.4
25.5[33]
24.0[33]
5Ј-d[GTA G(Ac-3,5-dimethylphenyl)AA TTC TAC] –3Ј 13i 8.2
5Ј-d[GTA G(2-fluorenyl)AA TTC TAC] –3Ј 13j
5Ј-d[GTA G(Ac-2-fluorenyl)AA TTC TAC] –3Ј 13k
5Ј-d[GTA G(4-biphenyl)AA TTC TAC] –3Ј 13l
5Ј-d[GTA G(Ac-4-biphenyl)AA TTC TAC] –3Ј 13m
5Ј-d[G(phenyl)TA GAA TTC TAC] –3Ј 13n
5Ј-d[G(Ac-phenyl)TA GAA TTC TAC] –3Ј 13o
5Ј-d[G(2-fluorenyl)TA GAA TTC TAC] –3Ј 13p
5Ј-d[G(Ac-2-fluorenyl)TA GAA TTC TAC] –3Ј 13q
5Ј-d[G(biphenyl)TA GAA TTC TAC] –3Ј 13r
5Ј-d[G(Ac-biphenyl)TA GAA TTC TAC] –3Ј 13s
23.3[33]
11.4
23.9[33]
7.9
34.5
23.8
32.5
In addition, the circular dichroism (CD) spectra were
measured for all oligonucleotides in their hybridized form.
CD spectra were recorded to prove the overall B-type-DNA
conformation of the lesion-containing DNA hybrids and
the unmodified reference duplex. For EcoRI-sequence
27.0
30.9[33]
26.2
[a] Conditions: 2 nmol EcoRI-oligonucleotide, 10 mm phosphate-
buffer pH 7.2, 140 mm NaCl, 1.0 mm EDTA.
Table 3. Tm values of 20mer-oligonucleotides 14a–m.[a]
Oligonucleotide
Tm [°C]
5Ј-d(ACA TGA GCA TCT ACG ACG CG) –3Ј 14a
68.5
63.1
56.3
61.0
55.4
61.1
5Ј-d[ACA TG(phenyl)A GCA TCT ACG ACG CG] –3Ј 14b
5Ј-d[ACA TG(Ac-phenyl)A GCA TCT ACG ACG CG] –3Ј 14c
5Ј-d[ACA TG(4-methylphenyl)A GCA TCT ACG ACG CG] –3Ј 14d
5Ј-d[ACA TG(Ac-4-methylphenyl)A GCA TCT ACG ACG CG] –3Ј 14e
5Ј-d[ACA TG(4-methoxyphenyl)A GCA TCT ACG ACG CG] –3Ј 14f
5Ј-d[ACA TG(Ac-4-methoxyphenyl)A GCA TCT ACG ACG CG] –3Ј 14g
5Ј-d[ACA TG(3,5-dimethylphenyl)A GCA TCT ACG ACG CG] –3Ј 14h
5Ј-d[ACA TG(Ac-3,5-dimethylphenyl)A GCA TCT ACG ACG CG] –3Ј 14i
5Ј-d[ACA TG(2-fluorenyl)A GCA TCT ACG ACG CG] –3Ј 14j
5Ј-d[ACA TG(Ac-2-fluorenyl)A GCA TCT ACG ACG CG] –3Ј 14k
5Ј-d[ACA TG(biphenyl)A GCA TCT ACG ACG CG] –3Ј 14l
66.6
62.3
64.7
63.4
62.7
64.0
63.5
5Ј-d[ACA TG(Ac-biphenyl)A GCA TCT ACG ACG CG] –3Ј 14m
[a] Conditions: 1 nmol 20mer-oligonucleotide, 1 nmol complementary oligonucleotide strand, 10 mm phosphate-buffer pH 7.2, 140 mm
NaCl, 1.0 mm EDTA.
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13n–q no conformational difference between the unmodi- h at 23 °C was determined. The cleavage rates were calcu-
fied oligonucleotide, the acetylated and non-acetylated lated as described previously.[34]
modified oligonucleotides was observed and in all cases a
By using oligonucleotides 13b–m, no restriction of any of
positive Cotton-effect was observed at 278 nm and a nega- these lesion-bearing oligonucleotides was detected. As an
tive one, which is less pronounced, at 250 nm typically example, incubation of 13m [modification within the
found for B-type-DNA structures (Figure 2).
EcoRI-cleavage site: (acetylamino)biphenyl] for 24 h, as
shown in Figure 3, clearly proves that no conversion of the
original oligonucleotide occurred. Thus, the 8-arylamino-
and 8-(acetylarylamino)-dG lesions within the cleavage site
caused a conformational distortion to such an extent that
the enzyme is unable to bind and/or cleave the DNA
double-strand. A modification introduced by a mono- or
polycyclic aromatic amine outside the cleavage site generally
led to cleavage of the hybrid although usually an increase
in the half-life for the restriction assay was detected. For
polycyclic 8-arylamino-dG damage, a half-life of 3.4 h was
found whereas damage caused by a monocyclic arylamine
led surprisingly to a higher half-life (6.5 h) and lower cleav-
age rates. In contrast, the cleavage of N-acetylated aniline
lesion 13o showed the same half-life as the reference hybrid
whereas N-acetylated dG-lesions 13p and 13s showed oppo-
site and markedly higher stability against restriction al-
though the damage was not placed within the cleavage site.
In the latter cases, it seems that the lesions introduced by
polycyclic arylamines 13p and 13s caused more significant
structural changes, which may result in weaker binding of
Figure 2. CD spectra of EcoRI-oligonucleotides 13a,n–q.
The shifts of the maxima in the CD spectra of modified
oligonucleotides 13n–q to higher wavelength are caused by
the (partly-) conjugated aromatic systems. In addition, no
overall conformational difference was observed for the
NarI-oligonucleotides and the 20mer duplexes.
EcoRI Restriction Assay
To study the effect on EcoRI endonuclease restriction of
8-(acetylarylamino)- or 8-(arylamino)-dG modified oligo-
nucleotides a previously used restriction assay was per-
formed.[33] As a control we have shown that the enzyme
EcoRI cleaved self-complementary, reference 12mer-oligo-
nucleotide 13a [5Ј-d(GTA GAA TTC TAC)-3Ј] into a 4mer-
(GTAG) and an 8mer-strand (AATTCTAC) using identical
Figure 3. HPLC-chromatogram of the EcoRI assay with 13m (see
assay conditions. For oligonucleotide 13a, a half-life of 2.5 Table 4; details are given in the Exp. Section).
Figure 4. HPLC-chromatogram of the EcoRI restriction assay with 13s (details are given in the Exp. Section).
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Synthesis and Effects of Specific-Site Damaged DNA Strands
the enzyme or less efficient recognition of the palindromic
hexamer (see 13r and 13s; Figure 4). In Table 4 the calcu-
lated half-lives are summarized. All values were calculated
according to a first-order reaction (t1/2 = ln2/k).
Table 4. Cleavage of damaged oligonucleotides 13 by nuclease
EcoRI.
Oligonucleotides
t1/2
5Ј-GTAGAATTCTAC-3Ј 13a
2.5
5Ј-d[GTA G(phenyl)AA TTC TAC] –3Ј 13b
5Ј-d[GTA G(Ac-phenyl)AA TTC TAC] –3Ј 13c
5Ј-d[GTA G(2-fluorenyl)AA TTC TAC] –3Ј 13j
5Ј-d[GTA G(Ac-2-fluorenyl)AA TTC TAC] –3Ј 13k
5Ј-d[GTA G(4-biphenyl)AA TTC TAC] –3Ј 13l
5Ј-d[GTA G(Ac-4-biphenyl)AA TTC TAC] –3Ј 13m
5Ј-d[G(phenyl)TA GAA TTC TAC] –3Ј 13n
5Ј-d[G(Ac-phenyl)TA GAA TTC TAC] –3Ј 13o
5Ј-d[G(Ac-2-fluorenyl)TA GAA TTC TAC] –3Ј 13p
5Ј-d[G(biphenyl)TA GAA TTC TAC] –3Ј 13r
5Ј-d[G(Ac-biphenyl)TA GAA TTC TAC] –3Ј 13s
no restriction
no restriction
no restriction
no restriction
no restriction
no restriction
6.5
2.5
4.4
3.4
5.6
Primer Extension Assay
As investigations of other DNA adducts have shown,
covalent DNA modifications significantly hamper the selec-
tivity and efficiency of DNA synthesis by replicative DNA
polymerases, whereas other DNA polymerases are effective
in performing lesion bypass beyond the site of damage.[35]
Thus, three DNA polymerases from different DNA poly-
merase families were used and studied for their effectiveness
in bypassing the 8-(acetylarylamino)-dG lesions. The ex-
periments were performed using standing-start conditions
with a 32P-labeled primer/template complex (Figure 5, a
and b). Single incorporations were examined to gain insight
into the impact of the lesion on selectivity, whereas ad-
ditional experiments employing all four dNTPs were used
to study the capability of a lesion bypass.
First, Pyrococcus furiosus (Pfu) DNA polymerase was
studied, which is a replicative DNA polymerase belonging
to sequence family B. In all cases of the 8-NAc-or the NH-
arylamine-dG lesions, no incorporation of the canonical or
the non-canonical nucleotides was observed proving the
high fidelity of DNA polymerase Pfu. This result confirmed
Figure 5. Effect of 8-NH- and 8-acetylarylamine-2Ј-dG lesions on
the primer extension by different DNA polymerases. (a) Used du-
plex for the primer extension assays in which G* points to the site
of modification. In the reference strand at this position an unmodi-
fied dG is present. (b) Gels obtained from the different primer ex-
tension assays using three different DNA polymerases (details are
given in the experimental part).
well the general opinion that non-acetylated and acetylated periment. In contrast, only in the case of NAc-phenyl- 14c
8-arylamine-dG adducts often lead to a slowing or stopping the full-length oligonucleotide was formed, whereas for
of the replication process.[36,37]
NH-phenyl-dG lesion 14b the full-length oligonucleotide as
Next, the highly processive, thermostable DNA polymer- well as a one or two nucleotide shorter product was ob-
ase FIREPol®, which is a member of the family A DNA served in addition to a strong kinetic hold after the nucleo-
polymerases and often serves for a wide range of PCR as- tide incorporation opposite the lesion. For both bulky
says was studied. The incorporation of a nucleotide oppo- aminobiphenyl lesions 14l and 14m, dCTP incorporation
site the DNA-lesion was successful and the canonical dCTP was observed exclusively but no elongation took place.
was preferably chosen in all cases. However, in the case of
As a member of the X family DNA polymerase involved
the NH-phenyl-dG lesion also misinsertion of the non-ca- in DNA repair, we investigated human DNA polymerase
nonical dATP was observed. Even more striking was that β.[38] For the 8-NAc-4-biphenyl- and the 8-NH-4-biphenyl
for the NAc-phenyl-dG lesion only the misinsertion of the lesions the canonical nucleotide dCMP was incorporated
non-canonical dATP was observed.
predominantly opposite to the modified dG, whereas in
If all 4 dNTPs were present, complete elongation to the cases of the two aniline-lesions again incorporation of the
full-length product (20 nt) was detected in the reference ex- non-canonical dAMP was observed to some extent. In the
Eur. J. Org. Chem. 2013, 1158–1169
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1163

S. Krüger, C. Meier
FULL PAPER
presence of all four dNTPs, complete elongation was ob- ages. Previously, also 8-(arylamino) adducts of dG or dA
served as expected. Interestingly, in the case of the lesion- showed the same behavior. However, irrespective of whether
containing oligonucleotides beside the full-length product a monocyclic or polycyclic arylamine DNA damages were in-
product that was one, two or more nucleotides shorter was vestigated, incorporation of the lesion outside to the EcoRI
observed. The DNA synthesis proceeded with less efficiency cleavage site only led to slower enzymatic cleavage. Surpris-
relative to the unmodified reference.
ingly, polycyclic acetylated DNA damages were found to
The ability to misincorporate a nucleotide significantly have a stronger influence than the corresponding monocy-
depended on the modification used as well as the modified clic acetylated DNA damages. Finally, it was shown that the
nucleobase. The use of a 20mer sequence with damaged ability to bypass a lesion within the DNA strand depended
dGs and three polymerases showed, with the Pfu DNA strongly on the investigated DNA polymerase. In most
polymerase, predominantly no incorporation of any nucleo- cases, the fidelity of the nucleotide incorporation opposite
tide owing to the high-fidelity of this polymerase. In these the dG-lesion was not hampered. Surprisingly, only the bor-
experiments human DNA polymerase β was able to bypass derline carcinogen aniline showed a different effect on the
the dG lesions and predominantly insert the canonical and replication. With two polymerases, a marked incorporation
also the non-canonical nucleotide opposite the lesion. The of dATP beside the canonical GTP was detected leading to
FIREPol® polymerase mainly showed the incorporation of a point mutation. The reason for this dCǞdA-misincorpo-
the canonical nucleotide opposite the dG lesions. Surpris- ration is still unknown. Further work to gain insights into
ingly, in the case of the NAc-phenyl modification, a selec- the caused structural changes within the DNA-duplex by
tive insertion of the non-canonical dA was observed. In our the 8-(arylamino)- and 8-(acetylarylamino) adducts is cur-
study the strong differences observed for family Y polymer- rently underway in our laboratories.
ases (e.g. yeast DNA polymerase η) for the acetylated and
the non-acetylated DNA lesions could not be observed. In
Experimental Section
those studies the bulky 2-(acetylamino)fluorene lesion led
to a complete stop in elongation, whereas in the case of the
2-aminofluorene lesion a slowing of the replication process
but a full elongation was observed.[39] In the case of the N-
acetylaniline lesion a full elongation was observed without
significant slowing of the replication.[40] This might be re-
lated to the family of the polymerase that was used in those
cases.
General Methods: All reactions were performed in glassware dried
with a heat gun and under a nitrogen atmosphere, except the syn-
theses of the hydroxylamines and hydroxamic acids. Commercial
solvents and reagents were used without further purification with
the following exceptions: pyridine, dichloromethane, and acetoni-
trile were distilled from calcium hydride; Dimethyl ether (DME)
and diethyl ether were dried with potassium and distilled under
1
nitrogen. Water was purified on a Milli-Q water system. H NMR
and 13C NMR spectra were recorded on AMX 400 MHz or DMX
500 MHz Bruker spectrometer. NMR spectra were reported rela-
tive to the NMR solvent peaks [1H NMR: δ = 2.50 (dimethyl sulf-
oxide; [D6]DMSO) and 7.16 (C6D6). 13C NMR: δ = 39.52 ([D6]-
DMSO) and 128.06 (C6D6)]. HRMS analysis was performed with
an analytical VG/70-250 F using FAB or Agilent Technologies,
6224 TOF LC/MS1200 spectrometer using ESI. IR spectra were
recorded on a Bruker Alpha P spectrophotometer operating in the
attenuated total reflection mode. Melting points were measured
with an apotec capillary melting point apparatus. Optical rotations
were measured on a Perkin–Elmer polarimeter by using a sodium
lamp operating at 598 nm and are reported as specific rotation.
Thin-layer chromatography was performed on aluminum sheets
coated with silica gel 60 F254 from Merck.
Conclusions
In this report, a modified experimental procedure for the
synthesis of 8-(acetylarylamino)-dG phosphoramidites is
described. These phosphoramidites were site-specifically in-
corporated into three different oligonucleotides. For the
NarI- and the self-complementary EcoRI sequence both the
thermal stabilities and CD-spectra were determined. In all
cases a marked decrease in the Tm values was detected for
the lesion-bearing DNA strands suggesting significant dis-
tortion of the double helix and resulting in lower hybrid
stability. In addition, the decrease was even stronger for the
8-(acetylarylamino) damage relative to the 8-(arylamino)
lesions. If this observation can be correlated to the detected
difference in syn/anti-preference of the glycosidic bond of
the dG adducts has to be proven in the future. Nevertheless,
no difference in the CD-spectra between the compounds
modified by an acetylated aromatic amine and those modi-
fied by a non-acetylated aromatic amine were measured.
The lesion-bearing 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 indicates a
marked local distortion of the DNA-hybrid at the modifica-
tion site that either blocks the cleavage of the double strand
or prevents a binding of the restriction enzyme. This effect
The synthesis of 3Ј,5Ј-bis-O-(tert-butyldimethylsilyl)-2Ј-deoxygu-
anosine and O6-(1H-benzotriazolyl)-3Ј,5Ј-bis-O-(tert-butyldimeth-
ylsilyl)-2Ј-deoxyguanosine were performed as described pre-
viously.[21,33]
General Procedure I. Arylhydroxylamines 5a–f: In a flask nitroaryl
compound (1 equiv.) was dissolved in ethanol/dichloroethane (1:1).
Raney nickel was added and the mixture was cooled to 0 °C. After
the addition of hydrazine hydrate (6.5 equiv.), the solvent was
stirred for 30–60 min and filtered. The solvent was removed in
vacuo and the product was recrystallized (toluene). Compounds 5
were isolated in 75–94% yields. Analytical data were identical to
those reported before.
General Procedure II. N-Aryl-N-acetylhydroxamic Acids 6a–f: In a
flask, N-arylhydroxylamine (1 equiv.) and saturated NaHCO3 solu-
was identical for all studied 8-(acetylarylamino)-dG dam- tion were suspended in Et2O and stirred at 0 °C. After the addition
1164
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Eur. J. Org. Chem. 2013, 1158–1169

Synthesis and Effects of Specific-Site Damaged DNA Strands
of AcCl (1.1 equiv.), the mixture was stirred at r.t. for 1 h. The
layers were separated and the organic layer was extracted with 1 m
NaOH (3ϫ). Then the aqueous layer was extracted with Et2O (3ϫ).
3 m HCl was added until the aqueous layer was acidic and extracted
with Et2O (3ϫ). The combined organic layers were dried (Na2SO4)
and the solvent removed in vacuo. Compounds 6 were isolated in
55–98% yields. Analytical data were identical to those reported be-
fore.[7,24]
8-[Acetyl(4-biphenylyl)amino]-3Ј,5Ј-bis-O-(tert-butyldimethylsilyl)-
2Ј-deoxyguanosine (7f): Following general procedure III, 4 (2.43 g,
3.97 mmol) and 6f (1.80 g, 7.93 mmol) gave product 7f as a slightly
brown solid (2.14 g, 3.04 mmol, 77%), m.p. 232–236 °C. [α]2D0 = +79
(c = 0.53, CHCl ). IR (neat): ν = 3668, 3312, 3150, 2954, 2928,
˜
3
2857, 1987, 1928, 1701, 1682, 1636, 1594, 1573, 1511, 1471, 1448,
1339, 1284, 1253, 1190, 1103, 1078, 1027, 1007, 970, 945, 881, 693,
632, 576, 555, 532, 502, 475, 464, 430, 403 cm–1 1H NMR
.
(400 MHz, [D6]DMSO): δ = 10.84 (s, 1 H), 7.71–7.67 (m, 4 H),
7.67–7.61 (m, 2 H), 7.51–7.42 (m, 2 H), 7.41–7.32 (m, 1 H), 6.40
(s, 2 H), 5.91–6.13 (m, 1 H), 4.69–4.42 (m, 1 H), 3.87–3.56 (m, 3
H), 3.16 (m, 2 H), 2.05 (s, 3 H), 0.85 (s, 9 H), 0.79 (s, 9 H), 0.08
(s, 6 H), –0.05 (s, 6 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ
= 169.5, 156.2, 139.5, 129.0, 127.6, 126.6, 126.4, 124.9, 87.3, 83.3,
72.2, 63.3, 36.0, 25.7, 25.6, 22.8, 5.4, 5.5, –4.8, –5.3 ppm. HRMS
(FAB): calcd. for C36H52N6O5Si2 [M + H] 705.3538; found
705.3612.
General Procedure III. 8-(Acetylarylamine) Adducts 7a–f: In a dried
flask compound 4 (1 equiv.), Cs2CO3 (2 equiv.) and N-arylacetylhy-
droxamic acid 6a–f (2 equiv.) were suspended in anhydrous DME
and stirred under a N2 atmosphere at room temperature. When the
reaction was complete the solvent was removed in vacuo. The prod-
uct was isolated by column chromatography (5% CH3OH/CH2Cl2).
8-(Acetylphenylamino)-3Ј,5Ј-bis-O-(tert-butyldimethylsilyl)-2Ј-de-
oxyguanosine (7a): Following general procedure III, 4 (5.00 g,
8.17 mmol) and 6a (10.2 g, 16.2 mmol) gave product 7a as a slightly
brown solid (3.58 g, 5.69 mmol, 70%). Analytical data were iden-
tical to those reported.[24]
General Procedure IV. Desilylation of C8-N-acetylarylamine Ad-
ducts 8a–f: Compound 7a–f was dissolved in tetrahydrofuran
(THF), then TBAF in THF (6 equiv.) and AcOH (12 equiv.) were
added simultaneously. When the reaction was complete the solvent
was removed in vacuo. The product was isolated by column
chromatography (10% CH3OH/CH2Cl2).
8-[Acetyl(4-methylphenyl)amino]-3Ј,5Ј-bis-O-(tert-butyldimethyl-
silyl)-2Ј-deoxyguanosine (7b): Following general procedure III, 4
(4.01 g, 6.55 mmol) and 6b (2.16 g, 13.1 mmol) gave product 7b as
a slightly brown solid (3.29 g, 5.12 mmol, 78%), m.p. 173–175 °C.
8-(Acetylphenylamino)-2Ј-deoxyguanosine (8a): Following general
procedure IV, 7a (1.00 g, 1.50 mmol) gave product 8a as a yellow
solid (568 mg, 1.42 mmol, 89%). Analytical data were identical to
those reported.[24]
[α]2D0 = +84 (c = 0.64, CHCl ). IR (neat): ν = 3151, 2928, 2856,
˜
3
1703, 1631, 1602, 1542, 1509, 1463, 1434, 1365, 1317, 1285, 1251,
1184, 1080, 1029, 956, 833, 812, 775, 729, 681, 601, 542, 506,
401 cm–1. 1H NMR (400 MHz, [D6]DMSO): δ = 10.82 (br. s, 1 H),
7.29–7.11 (m, 4 H), 6.39 (s, 2 H), 5.99–5.97 (m, 1 H), 4.53–4.46 (m,
1 H), 3.84–3.61 (m, 3 H), 3.17–3.16 (m, 1 H), 2.29 (s, 3 H), 2.10–
2.08 (m, 1 H), 1.99 (s, 3 H), 0.86 (s, 9 H), 0.81 (s, 9 H), 0.08 (s, 3
H), 0.06 (s, 3 H), –0.04 (s, 6 H) ppm. 13C NMR (101 MHz, [D6]-
DMSO): δ = 169.2, 156.2, 152.3, 138.3, 129.1, 128.9, 117.2, 113.1,
87.3, 87.0, 72.3, 63.1, 37.9, 25.7, 25.6, 20.5, 17.5, –4.8, –4.9, –5.4,
–5.5 ppm. HRMS (FAB): calcd. for C31H50N6O5Si2 [M + H]
643.3381; found 643.3454.
8-[Acetyl(4-methylphenyl)amino]-2Ј-deoxyguanosine (8b): Following
general procedure IV, 7b (3.28 g, 5.11 mmol) gave product 8b as a
yellow solid (2.04 g, 4.93 mmol, 96%), m.p. 174–175 °C. [α]2D0 = +84
(c = 0.64, CHCl ). IR (neat): ν = 3340, 3231, 1684, 1647, 1601,
˜
3
1568, 1540, 1510, 1474, 1396, 1369, 1320, 1286, 1176, 1102, 1038,
786, 712, 645, 581, 538, 474 cm–1 1H NMR (400 MHz, [D6]-
.
DMSO): δ = 11.51 (br. s, 1 H), 7.44–7.23 (m, 4 H), 6.73 (br. s, 2
H), 6.06–6.01 (m, 1 H), 5.31 (s, 1 H), 4.43–4.33 (m, 1 H), 3.92 (s,
1 H), 3.82–3.80 (m, 1 H), 3.66–3.48 (m, 3 H), 2.37–2.34 (m, 1 H),
2.30 (s, 3 H), 1.99 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO):
δ = 176.4, 156.3, 153.9, 149.1, 128.8, 125.1, 120.0, 88.1, 82.5, 71.2,
62.2, 48.6, 20.5, 20.2 ppm. HRMS (FAB): calcd. for C19H22N6O5
[M + H] 415.4652; found 415.1722.
8-[Acetyl(4-methoxyphenyl)amino]-3Ј,5Ј-bis-O-(tert-butyldimethyl-
silyl)-2Ј-deoxyguanosine (7c): Following general procedure III, 4
(845 mg, 1.38 mmol) and 6c (500 mg, 2.76 mmol) gave product 7c
as a slightly brown solid (649 mg, 0.99 mmol, 72 %), m.p. 155–
157 °C. [α]2D0 = +52 (c = 0.22, CHCl ). IR (neat): ν = 3145, 2952,
˜
3
8-[Acetyl(4-methoxyphenyl)amino]-2Ј-deoxyguanosine (8c): Follow-
ing general procedure IV, 7c (2.25 g, 3.41 mmol) gave product 8c
2928, 2855, 1702, 1632, 1606, 1569, 1541, 1508, 1462, 1435, 1365,
1316, 1287, 1246, 1181, 1105, 1081, 1060, 1029, 1006, 947, 830,
775, 731, 712, 682, 665, 626, 600, 552, 528, 402 cm–1. 1H NMR
(400 MHz, [D6]DMSO): δ = 10.82 (br. s, 1 H), 7.55–7.10 (m, 2 H),
6.94 (br. s, 2 H), 6.35 (s, 2 H), 6.01 (s, 1 H), 4.54 (s, 1 H), 3.88–
3.60 (m, 8 H), 1.97 (s, 3 H), 0.87 (s, 9 H), 0.81 (s, 9 H), 0.09 (br. s,
6 H), 0.04 (s, 6 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ =
176.2, 156.2, 153.1, 140.7, 131.0, 120.8, 115.0, 114.2, 87.3, 83.4,
72.3, 63.2, 55.3, 31.4, 25.7, 25.6, 23.7, 17.9, 17.6, –5.4, –5.5 ppm.
HRMS (FAB): calcd. for C31H50N6O6Si2 [M + H] 659.3330; found
659.3402.
as a white solid (1.31 g, 3.04 mmol, 89%), m.p. 155–156 °C. [α]2D0
=
+65 (c = 0.45, CHCl ). IR (neat): ν = 3317, 3155, 2931, 1676, 1630,
˜
3
1596, 1540, 1507, 1438, 1399, 1366, 1320, 1285, 1246, 1171, 1099,
1084, 1053, 1028, 987, 965, 946, 835, 814, 782, 728, 686, 600, 583,
1
542, 452 cm–1. H NMR (400 MHz, [D6]DMSO): δ = 10.85 (br. s,
1 H), 7.54–7.16 (m, 2 H), 6.98 (br. s, 2 H), 6.47 (br. s, 2 H), 6.07
(s, 1 H), 5.21 (br. s, 1 H), 4.94 (br. s, 1 H), 4.37 (s, 1 H), 3.83 (br.
s, 1 H), 3.76 (s, 3 H), 3.70–3.58 (m, 1 H), 3.57–3.46 (m, 1 H), 3.19–
3.13 (m, 2 H), 2.00 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]-
DMSO): δ = 172.7, 156.2, 153.5, 138.5, 128.0, 114.9, 125.0, 88.2,
85.2, 71.2, 62.1, 57.5, 23.1, 21.2 ppm. HRMS (FAB): calcd. for
C19H22N6O6 [M + H] 431.1601; found 431.1664.
8-[Acetyl(3,5-dimethylphenyl)amino]-3Ј,5Ј-bis-O-(tert-butyldimeth-
ylsilyl)-2Ј-deoxyguanosine (7d): Following general procedure III, 4
(5.01 g, 8.18 mmol) and 6d (2.96 g, 16.5 mmol) gave product 7d as
a slightly orange solid (4.88 g, 7.44 mmol, 91%). Analytical data
were identical to those reported.[24]
8-[Acetyl(3,5-dimethylphenyl)amino]-2Ј-deoxyguanosine (8d): Fol-
lowing general procedure IV, 7d (3.06 g, 4.66 mmol) gave product
8d as a white solid (1.77 g, 4.13 mmol, 89 %), m.p. 193–196 °C.
8-[Acetyl(2-fluorenyl)amino]-3Ј,5Ј-bis-O-(tert-butyldimethylsilyl)- [α]2D0 = +5 (c = 0.5, CH Cl /CH OH, 1:1; v/v). IR (neat): ν = 3325,
˜
2
2
3
2Ј-deoxyguanosine (7e): Following general procedure III, 4 (5.01 g,
8.18 mmol) and 6e (3.89 g, 16.2 mmol) gave product 7e as a slightly
brown solid (4.88 g, 7.77 mmol, 95%). Analytical data were iden-
tical to those reported.[24]
3148, 2923, 2874, 1677, 1632, 1592, 1539, 1434, 1398, 1366, 1303,
1280, 1260, 1100, 1084, 1053, 989, 966, 852, 835, 781, 715, 691,
1
674, 600, 561, 542, 450, 434, 403 cm–1. H NMR (400 MHz, [D6]-
DMSO): δ = 10.82 (br. s, 1 H), 6.96 (br. s, 3 H), 6.49 (br. s, 2 H),
Eur. J. Org. Chem. 2013, 1158–1169
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S. Krüger, C. Meier
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6.10–5.98 (m, 1 H), 5.03 (br. s, 2 H), 4.38 (s, 1 H), 3.83 (s, 1 H), 8-[Acetyl(4-methoxyphenyl)amino]-N2-[(dimethylamino)methylene]-
3.65 (dd, JHH = 11.5, JHH = 4.8 Hz, 1 H), 3.65 (dd, JHH = 11.7, 2Ј-deoxyguanosine (9c): Following general procedure V, 8c (1.30 g,
JHH = 5.1 Hz, 1 H), 3.20–3.11 (m, 2 H), 2.27 (s, 6 H), 2.00 (s, 3
H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ = 156.2, 153.5, 86%), m.p. 150–152 °C. [α]2D0 = +87 (c = 0.5, CH2Cl2/CH3OH; 1:1;
142.0, 88.1, 84.0, 83.9, 71.1, 62.1, 48.6, 22.5, 22.4, 20.8 ppm. v/v). IR (neat): ν = 3234, 2930, 1676, 1628, 1524, 1505, 1421, 1353,
3.02 mmol) gave product 9c as a yellow solid (1.26 g, 2.60 mmol,
˜
HRMS (FAB): calcd. for C20H24N6O5 [M + H] 429.1808; found
429.1477.
1320, 1283, 1243, 1170, 1108, 1052, 1026, 986, 967, 835, 785, 731,
580, 527, 480 cm–1. 1H NMR (400 MHz, [D6]DMSO): δ = 11.48
(br. s, 1 H), 8.47 (s, 1 H), 7.45 (s, 1 H), 7.27 (s, 1 H), 6.98 (s, 2 H),
6.13 (s, 1 H), 5.29 (s, 1 H), 4.90 (br. s, 1 H), 4.56–4.49 (m, 1 H),
3.85 (br. s, 1 H), 3.76 (s, 3 H), 3.70–3.61 (m, 1 H), 3.60–3.50 (m, 1
H), 3.18–3.14 (m, 2 H), 3.13 (s, 3 H), 3.03 (s, 3 H), 2.01 (s, 3
H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ = 168.3, 158.3,
157.1, 140.5, 138.6, 129.2, 128.0, 118.2, 99.5, 87.9, 70.9, 55.4, 48.4,
34.5, 22.3, 19.2 ppm. HRMS (FAB): calcd. for C22H27N7O6 [M +
H] 486.2023; found 486.2089.
8-[Acetyl(3,5-dimethylphenyl)amino]-N2-[(dimethylamino)methyl-
ene]-2Ј-deoxyguanosine (9d): Following general procedure V, 8d
(1.77 g, 4.13 mmol) gave product 9d as a yellow solid (1.66 g,
3.43 mmol, 83%), m.p. 176–177 °C. [α]2D0 = +44 (c = 0.31, CH2Cl2/
8-[Acetyl)(2-fluorenyl)amino]-2Ј-deoxyguanosine (8e): Following ge-
neral procedure IV, 7e (1.92 g, 2.69 mmol) gave product 8e as a
slightly brown solid (1.25 g, 2.54 mmol, 94 %), m.p. 198–19 °C.
[α]2D0 = +4.3 (c = 0.5, CH Cl /CH OH; 1:1; v/v). IR (neat): ν =
˜
2
2
3
3431, 3070, 2976, 2939, 2739, 2679, 2492, 1691, 1605, 1475, 1434,
1399, 1170, 1038, 739, 482 cm–1. 1H NMR (400 MHz, [D6]DMSO):
δ = 10.86 (s, 1 H), 7.94–7.89 (m, 2 H), 7.59–7.58 (m, 2 H), 7.40–
7.31 (m, 3 H), 6.44 (s, 2 H), 6.13 (s, 1 H), 5.23 (s, 1 H), 4.97 (s, 1
H), 4.40 (s, 1 H), 3.95 (s, 2 H), 3.85 (s, 1 H), 3.65–3.53 (m, 2 H),
3.17 (d, JHH = 5.1 Hz, 2 H), 2.06 (s, 3 H) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 161.8, 156.2, 153.4, 150.3, 143.3,
140.1, 127.0, 126.8, 125.1, 120.5, 120.2, 115.1, 88.1, 84.0, 71.1, 62.1,
40.6, 36.4, 22.7 ppm. HRMS (FAB): calcd. for C25H24N6O5 [M +
H] 489.1808; found 489.1873.
CH OH; 1:1; v/v). IR (neat): ν = 3307, 2926, 1679, 1629, 1524,
˜
3
1422, 1353, 1306, 1278, 1180, 1113, 1052, 988, 968, 853, 786, 720,
1
690, 663, 600, 574, 553, 534, 507, 465 cm–1. H NMR (400 MHz,
8-[Acetyl(4-biphenylyl)amino]-2Ј-deoxyguanosine (8f): Following ge-
neral procedure IV, 7f (1.40 g, 1.99 mmol) gave product 8f as a
yellow solid (1.31 g, 1.37 mmol, 69%), m.p. 90–92 °C. [α]2D0 = +48
[D6]DMSO): δ = 11.52 (s, 1 H), 8.47 (s, 1 H), 7.07–6.98 (m, 3 H),
6.10 (t, JHH = 3.7, JHH = 9.9 Hz, 1 H), 5.29 (d, JHH = 4.7 Hz, 1
H), 4.89–4.86 (m, 1 H), 4.45 (s, 1 H), 3.85–3.84 (m, 1 H), 3.69–3.64
(m, 1 H), 3.58–3.52 (m, 1 H), 3.14 (s, 3 H), 3.03 (s, 3 H), 2.98–2.93
(m, 1 H), 2.27 (s, 6 H), 2.01 (s, 3 H), 1.82–1.81 (m, 1 H) ppm. 13C
NMR (101 MHz, [D6]DMSO): δ = 175.6, 158.2, 149.5, 123.8,
123.1, 108.8, 87.8, 87.6, 71.6, 55.2, 40.7, 34.5, 22.4, 20.7, 18.7 ppm.
HRMS (FAB): calcd. for C23H29N7O5 [M + H] 484.2230; found
484.2303.
(c = 1.05, CH Cl /CH OH; 1:1; v/v). IR (neat): ν = 3675, 3304,
˜
2
2
3
2962, 2927, 2001, 1897, 1685, 1635, 1594, 1539, 1513, 1486, 1448,
1367, 1322, 1281, 1101, 1080, 1054, 1007, 965, 850, 765, 732, 693,
601, 442, 387 cm–1. 1H NMR (400 MHz, [D6]DMSO): δ = 10.73
(br. s, 1 H), 7.75–7.70 (m, 4 H), 7.69–7.65 (m, 2 H), 7.49–7.44 (m,
2 H), 7.40–7.34 (m, 1 H), 6.84 (s, 2 H), 6.10 (m, 1 H), 5.19 (br. s,
1 H), 4.80 (br. s, 1 H), 4.38 (s, 1 H), 3.83 (s, 1 H), 3.64 (dd, JHH
=
8-[Acetyl(2-fluorenyl)amino]-N2-[(dimethylamino)methylene]-2Ј-
deoxyguanosine (9e): Following general procedure V, 8e (2.05 g,
4.19 mmol) gave product 9e as a brown solid (2.05 g, 3.77 mmol,
90%). [α]2D0 = +59 (c = 0.22, CH2Cl2/CH3OH; 1:1; v/v). IR (neat):
10.9, JHH = 4.0 Hz, 1 H), 3.55–3.45 (m, 1 H), 2.97 (s, 1 H), 2.06
(s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ = 173.2, 156.6,
154.1, 150.4, 139.2, 129.0, 127.7, 127.5, 126.7, 125.5, 88.2, 84.0,
71.2, 62.1, 36.9, 22.3 ppm. HRMS (FAB): calcd. for C24H24N6O5
[M + H] 477.1808; found 477.1886.
ν = 3425, 2981, 1684, 1631, 1529, 1456, 1425, 1397, 1286, 1174,
˜
1
1115, 1037, 714, 474 cm–1. H NMR (400 MHz, [D6]DMSO): δ =
11.53 (s, 1 H), 8.49 (br. s, 1 H), 7.92–7.90 (m, 1 H), 7.80–7.76 (m,
1 H), 7.60–7.58 (m, 1 H), 7.41–7.31 (m, 4 H), 6.19 (br. s, 1 H), 5.39
(br. s, 1 H), 4.92 (br. s, 1 H), 4.51–4.44 (m, 1 H), 3.95 (s, 2 H), 3.87
(s, 1 H), 3.67 (s, 1 H), 3.56 (s, 1 H), 3.17–3.14 (m, 4 H), 3.03 (s, 6
H), 2.23 (s, 1 H), 2.07 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]-
DMSO): δ = 187.4, 172.3, 158.3, 157.1, 149.6, 143.4, 140.1, 136.1,
127.1, 126.8, 125.1, 123.9, 120.3, 87.9, 70.9, 61.9, 40.8, 37.5, 36.4,
26.5, 23.0 ppm. HRMS (FAB): calcd. for C28H29N7O5 [M + H]
544.2230; found 544.2295.
8-[Acetyl(4-biphenylyl)amino]-N2-[(dimethylamino)methylene]-2Ј-de-
oxyguanosine (9f): Following general procedure V, 8f (1.26 g,
1.31 mmol) gave product 9f as a brown solid (0.93 g, 0.93 mmol,
71%), m.p. 67–68 °C. [α]2D0 = +54 (c = 0.85, CH2Cl2/CH3OH; 1:1;
General Procedure V. N2-Formamidation of 8-(Acetylarylamine) Ad-
ducts 9a–f: In a dried flask, 8a–f (1 equiv.) and N,N-dimethylform-
amide diethyl acetal (2 equiv.) were suspended in anhydrous pyr-
idine and stirred under a N2 atmosphere at room temp. When the
reaction was complete the solvent was removed in vacuo. The prod-
uct was isolated by column chromatography (20 % CH3OH/
CH2Cl2).
8-(Acetylphenylamino)-N2-[(dimethylamino)methylene]-2Ј-deoxy-
guanosine (9a): Following general procedure V, 8a (2.04 g,
5.09 mmol) gave product 9a as a yellow solid (1.99 g, 4.37 mmol,
86%). Analytical data were identical to those reported.[24]
8-[Acetyl(4-methylphenyl)amino]-N2-[(dimethylamino)methylene]-
2Ј-deoxyguanosine (9b): Following general procedure V, 8b (2.58 g,
6.23 mmol) gave product 9b as a yellow solid (2.83 g, 6.04 mmol,
97%), m.p. 191–193 °C. [α]2D0 = +81 (c = 0.48, CHCl3). IR (neat):
v/v). IR (neat): ν = 3307, 3055, 3033, 2926, 2871, 2179, 1680, 1629,
˜
1523, 1485, 1422, 1354, 1323, 1283, 1178, 1113, 1054, 1031, 989,
846, 787, 766, 748, 732, 700, 575, 508, 447, 406 cm–1. 1H NMR
(400 MHz, [D6]DMSO): δ = 11.55 (s, 1 H), 8.49 (s, 1 H), 7.79–7.71
(m, 2 H), 7.71–7.64 (m, 2 H), 7.52–7.44 (m, 4 H), 7.41–7.33 (m, 1
H), 6.16 (s, 1 H), 5.30 (s, 1 H), 4.87 (s, 1 H), 4.47 (s, 1 H), 3.85 (s,
1 H), 3.72–3.60 (m, 1 H), 3.61–3.47 (m, 1 H), 3.14 (m, 2 H), 3.04
(s, 6 H), 2.07 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ =
173.1, 173.0, 158.3, 157.3, 149.6, 128.9, 127.6, 127.5, 126.7, 88.0,
87.9, 70.9, 61.9, 45.7, 40.7, 34.5 ppm. HRMS (FAB): calcd. for
C27H29N7O5 [M + H] 532.2230; found 532.2020.
ν = 3231, 2921, 1680, 1629, 1524, 1507, 1422, 1353, 1320, 1283,
˜
1180, 1112, 1052, 987, 968, 786, 727, 515 cm–1 1H NMR
.
(400 MHz, [D6]DMSO): δ = 11.53 (br. s, 1 H), 8.48 (s, 1 H), 7.40–
7.26 (m, 4 H), 6.13–6.12 (m, 1 H), 5.31–5.30 (m, 1 H), 4.88 (s, 1
H), 4.48–4.43 (m, 1 H), 3.87–3.83 (m, 1 H), 3.68–3.66 (m, 1 H),
3.57–3.56 (m, 1 H), 3.14 (s, 3 H), 3.04–3.01 (m, 4 H), 2.31 (s, 3 H),
2.15–2.13 (m, 1 H), 2.03 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]-
DMSO): δ = 174.2, 158.3, 157.1, 149.1, 129.3, 128.6, 127.6, 113.2,
87.9, 83.9, 70.9, 61.9, 40.8, 34.6, 20.5, 18.8 ppm. HRMS (FAB): General Procedure VI. 5Ј-O-Dimethoxytritylation of the 8-(Acetyl-
calcd. for C22H27N7O5 [M + H] 470.2074; found 470.2152. arylamine) Adducts 10a–f: In a dried flask, 9a–f (1 equiv.) was dis-
1166
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 1158–1169

Synthesis and Effects of Specific-Site Damaged DNA Strands
solved in anhydrous pyridine under nitrogen atmosphere and 4,4Ј-
dimethoxytrityl chloride (1 equiv.) was added. The mixture was
stirred at room temperature until the reaction was complete. The
solvent was removed in vacuo. The product was isolated by flash
chromatography on Alox (act. III; 5% CH3OH/CH2Cl2).
8-(Acetylphenylamino)-5Ј-O-dimethoxytrityl-N2-[(dimethylamino)-
methylene]-2Ј-deoxyguanosine (10a): Following general procedure
VI, 9a (200 mg, 0.44 mmol) gave product 10a as a slightly brown
solid (298 mg, 0.39 mmol, 89%), m.p. 111–113 °C. [α]2D0 = +21 (c =
[D6]DMSO): δ = 11.48 (s, 1 H), 8.20 (s, 1 H), 7.91–7.87 (m, 2 H),
7.63–7.52 (m, 2 H), 7.37–7.24 (m, 5 H), 7.13–7.12 (m, 7 H), 6.69–
6.61 (m, 4 H), 6.28 (br. s, 1 H), 5.74 (s, 1 H), 5.35 (s, 1 H), 4.63 (s,
1 H), 3.96–3.90 (m, 3 H), 3.68 (br. s, 7 H), 3.29–3.27 (m, 1 H),
3.13–3.06 (m, 1 H), 3.01 (br. s, 3 H), 2.95 (s, 3 H), 2.09 (s, 3 H) ppm.
13C NMR (101 MHz, [D6]DMSO): δ = 157.9, 157.8, 157.4, 157.2,
144.8, 144.0, 143.3, 140.1, 135.6, 127.5, 126.8, 126.4, 125.1, 120.2,
112.9, 112.8, 83.2, 54.9, 40.8, 36.4, 34.6 ppm. HRMS (FAB): calcd.
for C49H47N7O7 [M + H] 846.3537; found 846.3593.
8-[Acetyl(4-biphenylyl)amino]-5Ј-O-dimethoxytrityl-N2-[(dimeth-
ylamino)methylene]-2Ј-deoxyguanosine (10f): Following general pro-
cedure VI, 9f (0.93 g, 0.93 mmol) gave product 10f as a yellow solid
(600 mg, 0.39 mmol, 42%), m.p. 150–151 °C. [α]2D0 = +43 (c = 1.1,
0.1, CHCl ). IR (neat): ν = 2931, 1734, 1653, 1594, 1569, 1491,
˜
3
1436, 1357, 1286, 1055, 844 cm– 1 1 H NMR (400 MHz,
.
[D6]DMSO): δ = 11.48 (s, 1 H), 8.19 (s, 1 H), 7.41–7.11 (m, 14 H),
6.74–6.67 (m, 5 H), 6.23–6.17 (m, 1 H), 5.39–5.31 (m, 1 H), 4.62–
4.58 (m, 1 H), 3.94–3.89 (m, 1 H), 3.70–3.68 (m, 8 H), 3.29–3.23
(m, 1 H), 3.08–3.01 (m, 1 H), 3.00 (s, 3 H), 2.94 (s, 3 H), 2.04 (s, 3
H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ = 157.8, 157.4,
144.8, 129.4, 129.2, 127.5, 126.4, 112.7, 85.7, 83.0, 70.7, 54.8, 48.5,
34.5, 22.5 ppm. HRMS (FAB): calcd. for C42H43N7O7 [M + H]
758.3224; found 758.3336.
8-[Acetyl(4-methylphenyl)amino]-5Ј-O-dimethoxytrityl-N2-[(di-
methylamino)methylene]-2Ј-deoxyguanosine (10b): Following gene-
ral procedure VI, 9b (2.10 g, 4.64 mmol) gave product 10b as a
slightly brown solid (3.22 g, 4.17 mmol, 90%). Analytical data were
identical to those reported.[24]
CHCl ). IR (neat): ν = 3033, 2978, 2945, 2739, 2600, 2530, 2496,
˜
3
2179, 1682, 1629, 1526, 1507, 1481, 1443, 1423, 1397, 1286, 1247,
1172, 1113, 1072, 1033, 829, 807, 787, 766, 729, 699, 582, 551, 512,
462 cm–1. 1H NMR (400 MHz, [D6]DMSO): δ = 11.22 (s, 1 H),
8.10 (s, 1 H), 7.58–7.49 (m, 6 H), 7.36–7.32 (m, 3 H), 7.27–7.24 (m,
3 H), 7.14–7.12 (m, 3 H), 7.06–7.01 (m, 7 H), 6.14–6.10 (m, 1 H),
5.23 (s, 1 H), 4.50 (s, 1 H), 3.82 (s, 1 H), 3.62–3.60 (s, 1 H), 3.56
(s, 6 H), 3.07–3.03 (m, 1 H), 3.02–2.98 (m, 1 H), 2.90 (s, 3 H), 2.84
(s, 3 H), 1.97 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ =
180.4, 169.0, 157.9, 157.8, 157.5, 157.2, 144.9, 139.2, 135.6, 129.5,
129.4, 129.0, 127.6, 126.7, 126.5, 125.5, 112.9, 85.8, 85.2, 55.0, 54.9,
48.6, 45.3, 40.8, 34.7 ppm. HRMS (FAB): calcd. for C48H47N7O7
[M + H] 834.3537; found 834.3607.
8-[Acetyl(4-methoxyphenyl)amino]-5Ј-O-dimethoxytrityl-N2-[(di-
methylamino)methylene]-2Ј-deoxyguanosine (10c): Following gene-
ral procedure VI, 9c (1.24 g, 2.55 mmol) gave product 10c as a
slightly brown solid (1.31 g, 1.66 mmol, 65%), m.p. 159–160 °C.
General Procedure VII. 3Ј-O-Phosphitylation of the 8-(Acetylaryl-
amine) Adducts 11a–f: In a dried flask, 10a–f (1 equiv.) was dis-
solved in anhydrous CH2Cl2 and acetonitrile (1:1 v/v) under an
atmosphere of nitrogen and 4,5-dicyanoimidazole (DCI; 1 equiv.)
were added. The reaction mixture was treated with 2-cyanoethyl
bis(diisopropylamino)phosphate (1.5 equiv.). The mixture was
stirred at room temperature until the reaction was complete. The
solvent was removed in vacuo. The product was isolated by flash
chromatography on Alox (act. III); 1 % CH3OH/CH2Cl2). The
product was dissolved in benzene and gave the desired product as
a colorless solid after freeze-drying.
[α]2D0 = +24 (c = 0.1, CHCl ). IR (neat): ν = 3234, 2930, 2836, 1681,
˜
3
1627, 1607, 1526, 1505, 1463, 1422, 1344, 1322, 1288, 1244, 1173,
1
1111, 1078, 1027, 986, 827, 786, 754, 726, 701, 581, 527 cm–1. H
NMR (400 MHz, [D6]DMSO): δ = 11.36 (br. s, 1 H), 8.19 (s, 1 H),
7.34–7.22 (m, 9 H), 7.01–6.95 (m, 4 H), 6.78–6.69 (m, 4 H), 6.22–
6.16 (m, 1 H), 5.32 (br. s, 1 H), 4.15–4.05 (m, 1 H), 3.97–3.91 (m,
1 H), 3.77 (s, 3 H), 3.74–3.66 (m, 8 H), 3.17 (br. s, 2 H), 3.01 (s, 3
H), 3.00 (s, 3 H), 1.99 (s, 3 H) ppm. 13C NMR (101 MHz,
[D6]DMSO): δ = 157.9, 157.7, 157.2, 149.6, 135.6, 129.5, 127.5,
123.9, 112.9, 87.3, 85.2, 55.3, 55.4, 54.9, 40.0, 34.6, 15.3 ppm.
HRMS (FAB): calcd. for C43H45N7O8 [M + H] 788.3330; found
788.3393.
8-[Acetyl(3,5-dimethylphenyl)amino]-5Ј-O-dimethoxytrityl-N2-[(di-
methylamino)methylene]-2Ј-deoxyguanosine (10d): Following gene-
ral procedure VI, 9d (680 mg, 1.40 mmol) gave product 10d as a
yellow solid (1.01 g, 1.29 mmol, 92%), m.p. 110–112 °C. [α]2D0 = +37
2-Cyanoethyl N,N-Diisopropyl-8-(acetylphenylamino)-5Ј-O-dimeth-
oxytrityl-N2-[(dimethylamino)methylene]-2Ј-deoxyguanosine-3Ј-
phosphoramidite (11a): Following general procedure VII, 10a
(250 mg, 0.33 mmol) gave product 11a as a white solid (269 mg,
0.28 mmol, 85 %). Analytical data were identical to those re-
ported.[24]
Phosphoramidite 11b: Following general procedure VII, 10b
(500 mg, 0.65 mmol) gave product 11b as a white solid (521 mg,
0.54 mmol, 83%), m.p. 124–126 °C. [α]2D0 = +36 (c = 0.1, CHCl3).
(c = 0.5, CH Cl /CH OH, 1:1 v/v). IR (neat): ν = 2915, 1680, 1629,
˜
2
2
3
1609, 1527, 1507, 1423, 1302, 1247, 1175, 1112, 1076, 962, 827,
1
787, 754, 725, 582, 552 cm–1. H NMR (400 MHz, [D6]DMSO): δ
IR (neat): ν = 3056, 2998, 2932, 2835, 1685, 1629, 1606, 1580, 1528,
˜
= 11.39 (br. s, 1 H), 8.18 (s, 1 H), 7.27–6.95 (m, 12 H), 6.70–6.59
(m, 4 H), 6.20 (s,1 H), 5.33 (s, 1 H), 3.97 (s, 1 H), 3.69–3.67 (m, 6
H), 3.33 (s, 1 H), 3.07 (s, 1 H), 3.01 (s, 3 H), 2.94 (s, 4 H), 2.25–2.22
(m, 7 H), 2.04 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO): δ
= 157.8, 157.3, 157.1, 144.9, 135.6, 129.6, 129.5, 129.2, 127.6, 127.5,
127.4, 126.4, 118.1, 112.8, 112.7, 85.1, 83.1, 54.9, 54.8, 48.5, 40.7,
34.5, 20.7 ppm. HRMS (FAB): calcd. for C44H47N7O7 [M + H]
786.3537; found 786.3606.
1506, 1462, 1440, 1423, 1345, 1294, 1244, 1173, 1112, 1078, 1029,
901, 826, 789, 753, 726, 700, 636, 582, 523 cm–1 1H NMR
.
(400 MHz, [D6]benzene): δ = 11.35 (br. s, 1 H), 8.50–8.47 (m, 1 H),
7.78–7.76 (m, 4 H), 7.61–7.59 (m, 4 H), 7.32–7.30 (m, 2 H), 7.25–
7.22 (m, 1 H), 7.13–7.11 (m, 2 H), 6.89–6.88 (m, 4 H), 6.55 (br. s,
1 H), 5.43 (br. s, 1 H), 4.56 (br. s, 1 H), 3.83–3.78 (m, 2 H), 3.68
(br. s, 2 H), 3.61–3.59 (m, 7 H), 3.44–3.43 (m, 1 H), 3.05–3.01 (m,
3 H), 2.90–2.88 (m, 3 H), 2.74 (br. s, 3 H), 2.18 (s, 3 H), 1.53–1.49
(m, 4 H), 1.32–1.30 (m, 6 H), 1.25–1.24 (m, 6 H) ppm. 13C NMR
(101 MHz, [D6]benzene): δ = 159.1, 159.0, 158.0, 145.8, 136.4,
136.3, 130.6, 130.4, 129.5, 127.0, 118.9, 116.8, 113.4, 86.7, 58.6,
57.7, 55.0, 54.9, 54.8, 46.4, 43.8, 43.7, 43.5, 35.0, 24.6, 23.9, 21.2,
8-[Acetyl(2-fluorenyl)amino]-5Ј-O-dimethoxytrityl-N2-[(dimethyl-
amino)methylene]-2Ј-deoxyguanosine (10e): Following general pro-
cedure VI, 9e (2.00 g, 3.77 mmol) gave product 10e as a slightly
brown solid (2.50 g, 2.96 mmol, 79%), m.p. 180–181 °C. [α]2D0
=
+195 (c = 0.3, CH Cl /CH OH, 1:1 v/v). IR (neat): ν = 2982, 1681, 20.4, 19.9, 13.8 ppm. 31P NMR (162 MHz, [D6]benzene): δ = 162.8,
˜
2
2
3
1628, 1525, 1505, 1454, 1422, 1397, 1285, 1244, 1174, 1111, 1076,
162.4 ppm. HRMS (FAB): calcd. for C52H62N9O8P [M] 971.4459;
found 971.4546.
1028, 826, 786, 768, 735, 700, 580 cm–1 1H NMR (400 MHz,
.
Eur. J. Org. Chem. 2013, 1158–1169
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1167

S. Krüger, C. Meier
FULL PAPER
Phosphoramidite 11c: Following general procedure VII, 10c (1.29 g,
1.64 mmol) gave product 11c as a white solid (568 mg, 0.58 mmol,
35%), m.p. 142–143 °C. [α]2D0 = +74 (c = 0.06, CHCl3). IR (neat):
40.5, 34.6, 24.5, 24.3, 19.9 ppm. 31P NMR (162 MHz, [D6]-
benzene):
δ = 162.7, 162.4 ppm. HRMS (FAB): calcd. for
C57H64N9O8P [M + H] 1034.4615; found 1034.4658.
ν = 2963, 2930, 2836, 1683, 1628, 1607, 1527, 1506, 1462, 1442,
˜
Synthesis of the Oligonucleotides: Oligonucleotides were synthe-
sized on a 1 μmol scale using bz-protected dA, form-protected dG,
bz-protected dC and T phosphoramidites on a 394 DNA synthe-
sizer (Applied Biosystems) using phosphoramidites and solid sup-
ports purchased from ChemGenes. The manufacturerЈs standard
synthesis protocol was used, except that at the incorporation posi-
tion of the modified phosphoramidites the coupling was repeated
three times, each for 500 s. The oligonucleotides were purified by
HPLC using triethylammonium acetate buffer (pH 6.9; solvent 1)
and acetonitrile (solvent 2) on a C-18 reversed-phase column with
UV-detection. 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.
1423, 1345, 1325, 1288, 1246, 1200, 1175, 1112, 1075, 1029, 976,
898, 828, 787, 755, 726, 702, 683, 638, 582, 525 cm–1. 1H NMR
(400 MHz, [D6]benzene): δ = 10.83 (s, 1 H), 8.25 (s, 1 H), 7.44–
7.34 (m, 4 H), 7.13–6.55 (m, 13 H), 6.39 (s, 1 H), 5.32–5.15 (m, 1
H), 4.50–4.30 (m, 1 H), 3.68–3.13 (m, 16 H), 1.89 (s, 3 H), 1.16–
0.99 (m, 12 H) ppm. 13C NMR (101 MHz, [D6]benzene): δ = 159.1,
146.1, 146.0, 136.5, 136.2, 130.6, 130.4, 129.4, 127.0, 113.5, 113.6,
58.0, 55.0, 54.9, 46.6, 45.3, 43.9, 43.7, 43.6, 34.9, 24.6, 22.8, 19.5,
19.4, 19.1 ppm. 31P NMR (162 MHz, [D6]benzene): δ = 162.6,
162.4 ppm. HRMS (FAB): calcd. for C52H62N9O9P [M + Na]
1010.4408; found 1010.4294.
Phosphoramidite 11d: Following general procedure VII, 10d
(370 mg, 0.47 mmol) gave product 11d as a white solid (346 mg,
0.35 mmol, 74%), m.p. 127–129 °C. [α]2D0 = +91 (c = 0.1, CHCl3).
HPLC-ESI-MS for 12a: m/z calcd. for 3582.4; found 1189.3 (–3)
HPLC-ESI-MS for 12b: m/z calcd. for 3673.5; found 1835.7 (–2)
HPLC-ESI-MS for 12c: m/z calcd. for 3715.5; found 1856.3 (–2)
HPLC-ESI-MS for 12d: m/z calcd. for 3761.5; found 1879.7 (–2)
HPLC-ESI-MS for 12e: m/z calcd. for 3803.5; found 1900.4 (–2)
HPLC-ESI-MS for 12f: m/z calcd. for 3673.5; found 1835.8 (–2)
HPLC-ESI-MS for 12g: m/z calcd. for 3715.5; found 1856.8 (–2)
HPLC-ESI-MS for 12h: m/z calcd. for 3761.5; found 1879.8 (–2)
HPLC-ESI-MS for 12i: m/z calcd. for 3803.5; found 1900.4 (–2)
HPLC-ESI-MS for 13a: m/z calcd. for 3644.5; found 1214.8 (–3)
HPLC-ESI-MS for 13c: m/z calcd. for 3777.6; found 1887.4 (–2)
HPLC-ESI-MS for 13e: m/z calcd. for 3791.6; found 1894.4 (–2)
HPLC-ESI-MS for 13g: m/z calcd. for 3807.6; found 1902.2 (–2)
HPLC-ESI-MS for 13i: m/z calcd. for 3805.6; found 1904.4 (–2)
HPLC-ESI-MS for 13k: m/z calcd. for 3865.6; found 1931.3 (–2)
HPLC-ESI-MS for 13m: m/z calcd. for 3853.6; found 1925.4 (–2)
HPLC-ESI-MS for 13n: m/z calcd. for 3735.6; found 1866.8 (–2)
HPLC-ESI-MS for 13o: m/z calcd. for 3777.6; found 1887.8 (–2)
HPLC-ESI-MS for 13p: m/z calcd. for 3823.6; found 1910.3 (–2)
HPLC-ESI-MS for 13q: m/z calcd. for 3865.6; found 1913.3 (–2)
HPLC-ESI-MS for 13s: m/z calcd. for 3853.6; found 1925.9 (–2)
HPLC-ESI-MS for 14b: m/z calcd. for 6202.1; found 2066.4 (–3)
HPLC-ESI-MS for 14c: m/z calcd. for 6244.1; found 2080.4 (–3)
HPLC-ESI-MS for 14d: m/z calcd. for 6216.1; found 2071.1 (–3)
HPLC-ESI-MS for 14e: m/z calcd. for 6258.1; found 2085.1 (–3)
HPLC-ESI-MS for 14f: m/z calcd. for 6232.1; found 2076.4 (–3)
HPLC-ESI-MS for 14g: m/z calcd. for 6274.1; found 2090.4 (–3)
HPLC-ESI-MS for 14h: m/z calcd. for 6230.1; found 2075.7 (–3)
HPLC-ESI-MS for 14i: m/z calcd. for 6272.1; found 2089.4 (–3)
HPLC-ESI-MS for 14j: m/z calcd. for 6290.1; found 2095.4 (–3)
HPLC-ESI-MS for 14k: m/z calcd. for 6332.1; found 2109.7 (–3)
HPLC-ESI-MS for 14l: m/z calcd. for 6278.1; found 2091.44 (–3)
HPLC-ESI-MS for 14m: m/z calcd. for 6320.1; found 2105.7 (–3)
IR (neat): ν = 3236, 2964, 2930, 2837, 2248, 1682, 1629, 1608, 1527,
˜
1506, 1463, 1443, 1423, 1345, 1303, 1247, 1200, 1175, 1155, 1114,
1066, 1029, 976, 898, 878, 854, 827, 788, 754, 725, 701, 639, 581,
1
881, 519, 462, 396, 385 cm–1. H NMR (400 MHz, [D6]benzene): δ
= 10.77 (s, 1 H), 8.26–8.18 (m, 1 H), 7.13–6.52 (m, 16 H), 6.40 (s,
1 H), 5.32–5.18 (m, 1 H), 4.42 (br. s, 1 H), 3.74–2.91 (m, 16 H),
2.71–2.58 (m, 4 H), 2.52 (s, 3 H), 2.03 (s, 3 H), 1.85–1.68 (m, 4 H),
1.12–0.96 (m, 12 H) ppm. 13C NMR (101 MHz, [D6]benzene): δ =
159.1, 157.8, 145.8, 143.4, 136.5, 136.4, 136.2, 130.7, 130.6, 130.3,
128.6, 127.0, 126.9, 117.6, 113.5, 113.4, 86.7, 86.6, 59.0, 58.9, 57.9,
55.0, 54.9, 43.8, 43.7, 43.6, 40.8, 34.9, 34.8, 24.7, 24.6, 24.5, 24.4,
21.2, 20.1, 20.0 ppm. 31P NMR (162 MHz, [D6]benzene): δ = 162.8,
162.7 ppm. HRMS (FAB): calcd. for C53H64N9O8P [M + H]
986.4615; found 986.4681.
Phosphoramidite 11e: Following general procedure VII, 10e
(0.39 mg, 0.46 mmol) gave product 11e as a white solid (317 mg,
0.30 mmol, 66%), m.p. 124–125 °C. [α]2D0 = +19 (c = 0.42, CHCl3).
IR (neat): ν = 2964, 1686, 1630, 1526, 1508, 1456, 1424, 1344, 1291,
˜
1249, 1177, 1114, 1076, 1031, 977, 899, 828, 788, 768, 735, 703,
1
643, 583, 519, 469, 424 cm–1. H NMR (400 MHz, [D6]benzene): δ
= 11.33 (s, 1 H), 8.31 (s, 1 H), 7.86–6.66 (m, 20 H), 6.44 (br. s, 1
H), 5.29–5.19 (m, 1 H), 4.41 (br. s, 1 H), 3.64–3.23 (m, 16 H), 2.87–
2.81 (m, 1 H), 2.73–2.72 (m, 6 H), 2.55–2.53 (m, 3 H), 1.10–1.01
(m, 12 H) ppm. 13C NMR (101 MHz, [D6]benzene): δ = 159.1,
159.0, 157.9, 157.8, 145.7, 145.6, 144.8, 144.0, 143.5, 141.2, 136.3,
136.2, 130.6, 130.4, 128.6, 128.4, 127.0, 125.3, 120.7, 120.3, 118.5,
117.7, 113.4, 59.0, 58.8, 57.8, 54.9, 46.0, 43.7, 43.5, 40.8, 37.0, 34.9,
24.6, 24.4, 21.2, 20.2, 20.0 ppm. 31P NMR (162 MHz, [D6]-
benzene):
δ = 147.4, 147.3 ppm. HRMS (FAB): calcd. for
C58H64N9O8P [M + H] 1046.4615; found 1046.4684.
Phosphoramidite 11f: Following general procedure VII, 10f
(0.56 mg, 0.36 mmol) gave product 11f as a white solid (186 mg,
0.18 mmol, 46%), m.p. 78–80 °C. [α]2D0 = +30 (c = 0.11, CH2Cl2/
CH OH). IR (neat): ν = 2965, 2929, 1685, 1628, 1608, 1526, 1508,
˜
3
1487, 1462, 1424, 1345, 1326, 1289, 1249, 1177, 1114, 1075, 1031,
978, 898, 828, 788, 764, 728, 700, 584, 551, 516 cm–1. 1H NMR
(400 MHz, [D6]benzene): δ = 11.24 (br. s, 1 H), 8.24 (s, 1 H), 7.73–
6.64 (m, 22 H), 6.38 (s, 1 H), 4.38 (s, 1 H), 3.64–3.54 (m, 1 H), 3.37
(s, 3 H), 3.35 (s, 3 H), 3.33–3.14 (m, 2 H), 2.89–2.70 (m, 2 H), 2.58
(dd, JHH = 21.5, JHH = 6.6 Hz, 1 H), 2.47–2.44 (m, 2 H), 2.38 (d,
JHH = 7.2 Hz, 1 H), 1.73 (dd, JHH = 13.4, JHH = 5.9 Hz, 1 H),
1.46 (t, JHH = 6.2 Hz, 1 H), 1.15–0.85 (m, 18 H) ppm. 13C NMR
(101 MHz, [D6]benzene): δ = 159.0, 145.5, 143.9, 143.2, 136.1,
130.5, 130.3, 125.1, 113.5, 113.3, 83.4, 58.9, 57.7, 54.8, 43.6, 43.4,
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Eur. J. Org. Chem. 2013, 1158–1169

Synthesis and Effects of Specific-Site Damaged DNA Strands
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1
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examples of ESI mass spectra of the oligonucleotides 12c,e, 13i,m,
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Received: July 24, 2012
Acknowledgments
The authors are grateful for financial support obtained from the
Deutsche Forschungsgemeinschaft (DFG) (ME 1161/9-1) and the
University of Hamburg.
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Published Online: January 10, 2013
Eur. J. Org. Chem. 2013, 1158–1169
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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