Synthesis of DNA-oligonucleotides damaged by arylamine-modified 2′-deoxyguanosine

Article abstract of DOI:10.1080/15257770701490738

C8-Arylamine-dG adducts bearing a labile N-formamidine group at the exocyclic amino function were converted into their corresponding 5′-O-DMTr-3′-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-

Full text of DOI:10.1080/15257770701490738

Nucleosides, Nucleotides, and Nucleic Acids, 26:705–708, 2007
Copyright ꢀ Taylor & Francis Group, LLC
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ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490738
SYNTHESIS OF DNA-OLIGONUCLEOTIDES DAMAGED BY
ARYLAMINE-MODIFIED 2ꢀ-DEOXYGUANOSINE
N. Bo¨ ge, Z. Szombati, and C. Meier
Department of Chemistry, Organic
2
Chemistry, Faculty of Science, University of Hamburg Hamburg, Germany
C8-Arylamine-dG adducts bearing a labile N-formamidine group at the exocyclic amino func-
2
tion were converted into their corresponding 5ꢁ-O-DMTr-3ꢁ-O-phosphoramidite-C8-arylamine-dG
derivatives. These compounds were used for the automated synthesis of site-specifically modified
oligonucleotides. These oligonucleotides were characterized by ESI-MS and enzymatic digestion and
studied for their CD properties and Tm values.
Keywords DNA-damage; aromatic amines; carcinogenicity; Pd-cross-coupling; C8-
adducts
INTRODUCTION
Exposure to chemical carcinogens can occur from environmental or
work conditions, diet, smoking, and endogenous processes. Poly- and
monocyclic aromatic amines belong to the class of chemical carcinogens
that are known to form covalent adducts with DNA. Chemically damaged
DNA (by electrophiles) may be the reason for the induction of chemical
carcinogenesis.[1] If these covalently bonded modifications are not repaired,
they might compromise the fidelity of DNA replication, leading to muta-
tions and possibly cancer.[2,3]
The arylamines require a metabolic activation leading to ultimate car-
cinogens, which are arylnitrenium ions. The predominant reaction of the
arylnitrenium ion is the C8-position of 2ꢁ-deoxyguanosine (dG) leading to
the corresponding adduct as the major product.[4,5] Our interest is related
to DNA-adducts of monocyclic aromatic amines that act as so-called bor-
derline carcinogens like aniline or 4-methylaniline which are often used as
pharmacophores and can be found in cigarette smoke.[6]
The authors thank the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the University
of Hamburg for financial support and WACKER Fine Chemicals (Burghausen, Germany) for gifts of
reagents.
Address correspondence to N. Bo¨ge, Department of Chemistry, Organic Chemistry, Faculty
of Science, University of Hamburg, Martin-Luther-King Platz 6, 20146 Hamburg, Germany. E-mail:
boege@chemie.uni-hamburg.de
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To study the biochemical effects and the structure of “damaged”
oligonucleotides a strategy for site-specific incorporation of dG-carcinogen
adducts is needed. So far, modified oligonucleotide strands have been pre-
pared by post-synthetic treatment of oligonucleotides with electrophilic am-
ination reagents. However, the yields were very low (1–5%).[7,8] Due to
the low yields, this procedure, therefore, is only applicable for oligonu-
cleotide sequences containing one dG. More suitable would be a strategy
for a site-specific incorporation of modified dG using the phosphoramidite
approach.
RESULTS
Recently, we published a highly efficient synthesis for the C8-arylamine-
dG adducts using palladium cross-coupling, their convertion into the cor-
responding phosphoramidites and the synthesis of site-specifically modified
oligonucleotides.[9] For DNA-synthesis a protecting group for the exocyclic
amino function is necessary. In the past we used the regular isoButyryl (iBu)
group. After oligonucleotide synthesis deprotection of the amino function
was done in concentrated ammonia solution within 16 hours at 55◦C. Be-
cause of the sensitivity of the arylamine-dG adducts to alkaline conditions
a much faster deprotection is preferable to increase the yields of the mod-
ified oligonucleotides. Therefore, the more labile formamidine protecting
group was introduced. Adducts of seven different aromatic amines (aniline,
4-methoxyaniline, 4-methylaniline, 4-cyanoaniline, 3,5-dimethylaniline, 4-
aminobiphenyl, and 2-aminofluorene) have been prepared in 60–91%
yield. Subsequently, the adducts were converted into the N 2-formamidine-
protected phosphoramidites. The synthesis is summarized in Figure 1.
Using the phosphoramidites a series of modified oligonucleotides bear-
ing C8-arylamine-dG adducts have been prepared using standard oligonu-
cleotide synthesis. For the incorporation of the modified phosphoramidites
three coupling cycles were used with a coupling time of 500 seconds. Cleav-
age of the oligonucleotides from the solid support (CPG) and deprotection
FIGURE 1 Synthesis of the arylamine-adduct phosphoramidites.

DNA-Oligonucleotides
707
was done with aqueous ammonia within 4 hours at 40◦C. β-Mercaptoethanol
was added to prevent a possible oxidative cleavage of the adducts under alka-
line conditions. Purification of the modified oligonucleotides has been per-
formed by HPLC following a DMTr-off protocol and a RP-C18-column using
triethylammonium acetate buffer (pH 6.9)/acetonitrile gradients. Due to
the change to the more labile protecting group the yields of the modified
oligonucleotides were 4- to 5-fold higher as compared to the iBu-route. The
oligonucleotides have been studied for purity by HPLC analysis and were
characterized by ESI-mass spectroscopy and enzymatic digestion.
Tm values of a self-complementary strand (5ꢁ-TATATCXATATA-3ꢁ and a
mixed 30mer-oligonucleotide (5ꢁ-AAA TXA ACC TAT CCT CCT TCA GGA
CCA ACG-3ꢁ) against the complementary strand were measured. The Tm
value of the unmodified self-complementary strand was found to be 33◦C.
A considerable decrease in the Tm was found when dG was replaced by an
arylamine-dG adduct: In the case of 4-methoxyaniline the Tmdrops to 19.1
◦C, with a 4-aminobiphenyl-adduct to 20.0◦C. In both cases a second Tm
was found 66–69◦C) probably a result of a hairpin structure.[9] The unmod-
ified 30mer-oligonucleotide showed a Tm value of 66◦C. For the modified
oligonucleotides the values differ from 63.5-64.1◦C. Again, one modification
was responsible for a loss of thermal stability of the duplex. Interestingly,
the effects of the strong carcinogens 4-aminobiphenyl and 2-aminofluorene
were found to be comparable to the borderline carcinogens.
Further structural effects of the incorporation of the adducts into
oligonucleotides were studied using CD-spectroscopy. However, no differ-
ence in the CD-spectra was observed for the oligonucleotides bearing the
adduct compared to the unmodified cases. All curves point to B-type DNA
duplexes.
REFERENCES
1. Garner, R.C. The role of DNA adducts in chemical carcinogenesis. Mutat. Res. 1998, 402, 67–75.
2. Neumann, H.G. The role of DNA damage in chemical carcinogenesis of aromatic amines. Cancer Res.
Clin. Oncol. 1986, 112, 100–106.
3. Beland, F.A.; Kadlubur, F.F. DNA adduct formation and mutation induction by nitropyrenes in
Salmonella and Chinese hamster ovary cells: relationships with nitroreduction and acetylation. Envir.
Health Perspect. 1985, 62, 19–23.
4. Turesky, R.J.; Rossi, S.C.; Welti, D.H.; Lay, J.O.; Kadlubar, F.F. Characterization of DNA
adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and
N-hydroxy-2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine.
Chem. Res. Toxicol. 1992, 5, 479–490.
5. Tureky, R.J.; Markovic, J. DNA Adduct Formation of the Food Carcinogen 2-Amino-3-
methylimidazo[4,5-f]quinoline at the C-8 and N2 Atoms of Guanine. Chem. Res. Toxicol. 1994, 7,
752–761.
6. Meier, C.; Boche, G. The modification of guanine nucleosides and nucleotides by the borderline
arylamine carcinogens 4-methyl- and 4-methoxyaniline: chemistry and structural characterization.
Carcinogenesis 1991, 12, 1633–1640.

708
N. Bo¨ge et al.
7. Shibutani, S.; Fernades, 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.
8. Marques, M.M.; Mourato, L.L.; Santos, M.A.; Beland, F.A. Synthesis, characterization, and conforma-
tional analysis of DNA adducts from methylated anilines present in tobacco smoke. Chem. Res. Toxicol
1996, 9, 99–108.
9. Meier, C.; Bo¨ge, N.; Gra¨sl, S. Synthesis and Properties of oligonucleotides containing C8-
deoxyguanosine arylamine-adducts of borderline carcinogens. J. Org. Chem. 2006, 71, 9728–9738.