Exocyclic deoxyadenosine adducts: from environmental carcinogens to antiviral drugs.
2010-07
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Exocyclic deoxyadenosine adducts: from environmental carcinogens to antiviral drugs.
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2010-07
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Abstract
1,2,3,4-Diepoxybutane (DEB) is considered the ultimate carcinogenic metabolite
of 1,3-butadiene, an important industrial chemical and environmental pollutant present in
urban air. Although it preferentially modifies guanine bases within DNA, DEB induces a
large number of A → T transversions, suggesting that it forms strongly mispairing lesions
at adenine nucleobases. Three potentially mispairing exocyclic adenine lesions of DEB,
N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (compound 2), 1,N6-(2-hydroxy-
3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (compound 3), and 1,N6-(1-
hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (compound 4), have been
recently identified in the present work. The structures and stereochemistry of the novel
DEB-dA adducts were determined by a combination of UV and NMR spectroscopy,
tandem mass spectrometry, and independent synthesis. We found that synthetic N6-(2-
hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine (compound 1) representing the product of
N6-adenine alkylation by DEB spontaneously cyclizes to form 3 under aqueous
conditions or 2 under anhydrous conditions in the presence of organic base. Compound 3
can be interconverted with 4 by a reversible unimolecular pericyclic reaction favoring 4
as a more thermodynamically stable product. Both 3 and 4 are present in double stranded
DNA treated with DEB in vitro and in liver DNA of laboratory mice exposed to 1,3-
butadiene by inhalation. We propose that in DNA under physiological conditions, DEB
alkylates the N-1 position of adenine in DNA to form N1-(2-hydroxy-3,4-epoxybut-1-yl)-
adenine adducts, which undergo an SN2-type intramolecular nucleophilic substitution and
rearrangement to give 3 (minor) and 4 (major). A post-synthetic methodology for
preparing DNA oligomers containing stereo- and site-specific 2 and 3 was developed in
v
order to investigate their biological properties. DNA oligomers containing site specific 6-
chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to
generate oligomers containing compound 1, followed by their spontaneous cyclization to
1,N6-γ-HMHP-dA lesions. N6,N6-DHB-dA containing strands were prepared analogously
by coupling 6-chloropurine containing DNA with 3S,4S or 3R,4R pyrrolidine-3,4-diols.
Oligodeoxynucleotide structures were confirmed by ESI- MS, exonuclease ladder
sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy
studies of DNA duplexes containing N6,N6-DHB-dA and 1,N6-γ-HMHP-dA revealed that
both lesions lower the thermodynamic stability of DNA when paired with dT. However,
the stability of 1,N6-γ-HMHP-dA containing DNA duplexes was greater when adenine
residue was placed opposite the lesion, suggesting that DEB-dA adducts preferentially
pair with A, potentially leading to A → T transversions during DNA replication. Solution
state NMR and site specific mutagenesis were conducted to further test the role of
exocyclic DEB-dA adducts in mutagenesis.
In the second part of this work, a series of purine and pyrimidine nucleoside
analogues modified at N6 and N4 positions by various substituted pyrrolidines and their
chain terminator analogues that lack a 3' hydroxyl group were synthesized as potential
inhibitors of HIV reverse trascriptase. Reverse transcriptase (RT) is one of the three
enzymes encoded by the Human Immunodeficiency Virus type 1 (HIV-1), the causative
agent of AIDS and one of the main targets for antiviral therapy. Nucleoside analogues
inhibiting the DNA polymerase activity of RT, dramatically reduced the HIV-1
proliferation. The synthetic strategies allow us to prepare most of the targeted compounds directly from the commercially available compounds. All compounds were tested against HIV-1 in CEM SS cells.
Description
University of Minnesota Ph.D. dissertation. July 2010. Major: Chemistry. Advisors: Dr. Natalia Tretyakova and Dr. Darrin M York. 1 computer file (PDF); xxvii, 367 pages.
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