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
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.
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.
Seneviratne, Uthpala Indrajith.
Exocyclic deoxyadenosine adducts: from environmental carcinogens to antiviral drugs..
Retrieved from the University of Minnesota Digital Conservancy,
Content distributed via the University of Minnesota's Digital Conservancy may be subject to additional license and use restrictions applied by the depositor.