Synthesis of oligonucleotides bearing an arylamine modification in the C8-position of 2′-deoxyguanosine

Article abstract of DOI:10.1081/NCN-200060267

C8-Arylamine-dG adducts were converted into their corresponding 5′-0-DMTr-3′-0-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. The oligonucleotides were stu

Full text of DOI:10.1081/NCN-200060267

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SYNTHESIS OF OLIGONUCLEOTIDES BEARING AN
ARYLAMINE MODIFICATION IN THE C8-POSITION OF 2′-
DEOXYGUANOSINE
a
a
b
b
C. Meier , S. Gräsl , I. Detmer & A. Marx
a
Institute of Organic Chemistry, University of Hamburg , Germany
b
Fachbereich Chemie, University of Konstanz , Konstanz, Germany
Published online: 15 Nov 2011.
To cite this article: C. Meier , S. Gräsl , I. Detmer & A. Marx (2005) SYNTHESIS OF OLIGONUCLEOTIDES BEARING AN
ARYLAMINE MODIFICATION IN THE C8-POSITION OF 2′-DEOXYGUANOSINE, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7,
691-694, DOI: 10.1081/NCN-200060267
To link to this article: http://dx.doi.org/10.1081/NCN-200060267
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):691–694, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060267
SYNTHESIS OF OLIGONUCLEOTIDES BEARING AN ARYLAMINE
MODIFICATION IN THE C8-POSITION OF 2’-DEOXYGUANOSINE
5
C. Meier and S. Gr ¨a sl
Germany
Institute of Organic Chemistry, University of Hamburg,
Fachbereich Chemie, University of Konstanz, Konstanz,
5
I. Detmer and A. Marx
Germany
5
C8-Arylamine-dG adducts were converted into their corresponding 5’-O-DMTr-3’-O-phosphorami-
dite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-
specifically modified oligonucleotides. The oligonucleotides were studied for their CD properties, T
m
values, and their effects on primer extension assays using human DNA-polymerase b.
Keywords DNA Damage, C8-Arylamine Adducts, Chemical Carcinogenesis, Mutations
INTRODUCTION
Instead of 2-aminofluorene, 1- or 2-naphthylamine, monocyclic aromatic
amines like anisidine belong to the class of borderline carcinogens. However, both
groups led after metabolic activation predominately to the formation of the same
type of covalent DNA-product: the C8-arylamine-2’-deoxyguanosine adduct (C8-
[
1]
ArNH-dG). The extent of DNA-damage from both classes of carcinogens is
comparable. If these DNA-damages are not repaired, they can compromise the
fidelity of DNA replication and cause mutations and possibly cancer. So far the
difference of the effects caused by strong carcinogens and the borderline
carcinogens on DNA-related processes is not known. To study the biochemical
effects, structure and repair of these adducts, a strategy for the site-specific
incorporation of dG-carcinogen adducts into oligonucleotides is needed. So far,
modified oligonucleotide strands have been prepared by post-synthetic treatment
[2]
with electrophilic amination reagents, resulting in very low amounts of the
[3]
oligonucleotides (1–5% yield). Due to the low yields, this procedure is therefore
only applicable for one dG containing oligonucleotide sequences. A better way
Address correspondence to C. Meier, Institute of Organic Chemistry, University of Hamburg, Martin-Luther-
King-Platz 6, Hamburg D-20146, Germany.
691
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692
C. Meier et al.
would be a strategy for a site-specific incorporation of the modified dG using the
phosphoramidite approach.
RESULTS
Recently we published a highly efficient synthetic protocol for the synthesis of
the C8-arylamine-dG adducts using a palladium-catalyzed Buchwald-Hartwig cross-
[
4]
coupling reaction. The heteroaromatic system should be appropriately protected
for the metal-catalyzed cross-coupling. Therefore, we used the electron-withdrawing
iso-butyryl-group at the exocyclic amino group and the benzyl or cyanophenylethyl
6
(
CPE) protection group at the O -position. Without the latter protecting group, the
cross-coupling was unsuccessful. Adducts of different aromatic and heteroaromatic
systems have been prepared in 60–80% yield. Subsequently, the adducts were
converted into the corresponding phosphoramidites. The synthesis is briefly
summarized in Figure 1.
Using these phosphoramidites, numerous oligonucleotides bearing one anilinyl-,
toluidinyl-, anisidinyl-, or aminobiphenyl-dG adduct have been prepared by
standard oligonucleotide synthesis on a CPG support. Three cycles of arylamine-
adduct phosphoramidite addition were used and the coupling periods were
extended to 60 s. Cleavage of the oligonucleotides from the solid support has been
done with aqueous ammonia in the presence of mercaptoethanol because these
adducts are base-labile. Purification of the adduct containing oligonucleotides has
been performed by HPLC using a DMTr-off protocol and a Waters X-Terra column.
The oligonucleotides have been studied for purity by HPLC analysis and were
characterized by MALDI-mass spectrometry.
Then, T values of the homo-thymidine oligonucleotides bearing one modified
m
dG in the center hybridized with oligo-dA strand bearing all four different bases
opposite to the modification site were determined. The T value of a reference
m
(
T) (dG)T Á (dA) (dC)(dA) duplex was found to be 39.4°C. A considerable
7
7
7
7
decrease in the T was found when the dG was replaced by the arylamine-adduct.
m
The highest T value for the adduct-bearing oligonucleotide was measured if a dC
m
FIGURE 1 Synthesis of the arylamine-adduct phosphoramidites.

C. Meier et al.
693
TABLE 1 T
m
Values of the T
7
(Arylamine-dG)T
7
Á dA
7
XdA
7
Hybrid
7 7
value (°C) against dA XdA
T
m
Oligonucleotide
X = C
X = G
X = T
X = A
TTTTTTT(dG)TTTTTTT
TTTTTTT(Tol-dG) TTTTTTT
TTTTTTT(ABP-dG) TTTTTTT
39.4
34.9
32.8
28.6
27.3
25.7
30.1
28.4
26.6
27.7
26.2
25.4
nucleotide was present in the opposite strand (T = 34.9°C and 32.8°C for the
m
toluidine and the aminobiphenyl-adduct, respectively). In the case of the toluidine
adduct, T values dropped further when dG or dA was placed opposite to the
m
adduct. Such an effect was also observed in the case of the reference oligo hybrid.
However, in the case of the aminobiphenyl adduct, the T was independent to the
m
nucleotide opposite to the adduct (Table 1).
Next, a mixed sequence 15-mer-oligonucleotide was used. Here, different
arylamine-adducts were incorporated in the middle while in the opposite strand
always dC was placed. The unmodified hybrid showed a T value of 51.6°C. T
m
m
values of the modified 15-mers were found to be 46.3°C (anilinyl-), 46.4°C
toluidinyl-), 45.0 (aminobiphenyl-), and 46.3°C (anisidinyl-adduct). Again, one
(
modification only was responsible for a considerable loss of thermal stability of the
duplex. Interestingly, the effect of the strong carcinogen aminobiphenyl was only
slightly stronger as compared to the borderline carcinogens aniline, toluidine,
and anisidine.
Further structural effects of the incorporation of the adducts into the
oligonucleotides were studied using CD-spectroscopy. However, no difference in
the CD-spectra was observed for the adduct-bearing oligonucleotides compared to
the unmodified case. All curves show shapes found previously for B-type DNA
duplexes. No difference between the aminobiphenyl adduct and the monocyclic
aromatic amines can be observed. The same CD properties have been found in
the case of the T (arylamine-dG)T Á dA XdA hybrids. Again, all showed typical
7
7
7
7
shapes for B-type DNA and no difference to the reference hybrid.
Finally, first experiments for primer-template extension studies have been
performed. As polymerase, the human enzyme pol b has been used. The adduct
has been incorporated into a 34-mer template strand. The primer ends directly
before the adduct. Thus, standing start conditions were used. Then, all four NTPs
were added separately. Only dCTP was incorporated opposite to the arylamine
adducts in all cases. However, a futher experiment was performed in which the
dC opposite to the arylamine adduct is already incorporated in the primer.
Thus, experiments under standing start + 1 conditions where done (Figure 2).
Surprisingly, in these experiments DNA pol b showed a considerable loss in fidelity!
Thus, errors seem to take place not at the opposite site of the adduct but one
step downstream. This effect was most apparent for the aminobiphenyl adduct but
was also observed in lower extent for the monocyclic aryl amines. This is an

694
C. Meier et al.
FIGURE 2 Standing start + 1 primer-extension experiment with DNA-polymerase b.
entirely unexpected result, which may have an important impact on the mutation
probability. Further experiments will be done to explain this new and unexpected
lack of fidelity in enzymatic DNA synthesis.
REFERENCES
1
.
Meier, C.; Boche, G. The modification of guanine nucleosides and nucleotides by the borderline carcinogens
4-methyl- and 4-methoxyaniline: chemistry and structural characterisation. Carcinogenesis 1991, 12(6), 1633–
1640.
2
3
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Beland, F.A.; Kadlubar, F.F. Formation and persistence of arylamine DNA adducts in vivo. Environ. Health
Perspect. 1985, 62, 19–36.
Shibutani, S.; Fernandes, A.; Suzuki, N.; Zhou, L.; Johnson, F.; Grollman, A.P. Mutagenesis of the
N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine DNA adduct in mammalian cells.
Sequence context effects. J. Biol. Chem. 1999, 274, 27433–27438.
4.
Meier, C.; Gr ¨a sl, S. Highly efficient synthesis of a phosphoramidite building block of C8-deoxyguanosine
adducts of aromatic amines. Synlett 2002, 5, 802–804.