Exposure to carcinogens
can occur from environmental or work conditions, diet, smoking and endogenous
processes. Poly- and monocyclic aromatic amines, e.g. aniline 1
, 4-aminobiphenyl 4
and 2-aminofluorene 6
(Figure 1), belong to the class of
chemical carcinogens that form covalently bound adducts to the DNA double helix.
Covalent damage of DNA (by electrophiles) may be one reason for the induction
of chemical carcinogenesis because if these DNA damages are not repaired, they
might compromise the fidelity of DNA replication which finally leads to
mutations and possibly cancer.
Aromatic amines belong to
the group of indirect carcinogens because they require a metabolic activation
leading to the so-called ultimate carcinogen (Scheme 1). The initial step is an
oxidation catalyzed by cytochrome P450 of the arylamine to the corresponding N
-hydroxylamine, followed by
esterification into an N
-acetyl transferase (NAT) or into
a sulfate by a sulfotransferase (ST) to give the ultimate carcinogens.
Borderline carcinogens and carcinogenic aromatic
The predominant reaction of the
arylnitrenium ion occurs at the C8-position of 2’-deoxyguanosine (dG) and,
although to a smaller amount, 2’-deoxyadenosine (dA), leading to C8-adducts 7, 8
and 9, 10
. Moreover, N2
of dG 11, 13
of dA 12
have been identified as
Metabolism of arylamines and resulting
We are working on synthetic
approaches to such nucleobase adducts using not only the borderline
carcinogens, but also poly- and heterocyclic aromatic amines.
step in the chemical synthesis for most of these DNA adducts is a Pd-catalyzed
C-N-bond formation reaction. Subsequently, these adducts were converted into
the corresponding phosphoramidites.
Figure 2. Key steps in the investigation of arylamine modified oligonucleotides
DNA polymerase enzymes employ a number of innate fidelity mechanisms to
ensure the faithful replication of the genome. However, when confronted with DNA
damage, their fidelity mechanisms can be evaded, resulting in a mutation that
may contribute to the carcinogenic process.
These modified oligonucleotides are able to block DNA
replication, occasionally bypassed and cause mutations if they are paired with
an incorrect base.
Figure 3. Effect of C8-arylamine-adducts on DNA polymerases
Figure 4: Cutting of the EcoRI-Sequence
To investigate the impact on
enzyme recognition of arylamine damaged oligonucleotides by an endonuclease,
the EcoRI restriction enzyme was chosen.
The nuclease EcoRI cleaves the
self-complementary, 12-mer oligonucleotide into a 4-mer (GTAG) and an
8-mer strand (AATTCTAC).
After site selective incorporation of various
arylamine damages into the self-complementary sequence, restriction assays were
performed and monitored by HPLC. The restriction was completely inhibited when
the damaged nucleotide was located within the cleavage site.
Figure 5: HPLC chromatogram of EcoRI restriction
with the DESY (german electron-synchrotron) we investigate the crystallographic
structure of our modified oligonucleotides on the basis of improvement of the
buffer, pH-value and different counterions.
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