Open Access Thesis
Date of Award
Dr. Lawrence J. Marnett
Malondiadehyde (MDA) is a known carcinogen and mutagen produced by lipid peroxidation and prostaglandin synthesis in vivo. MDA is unique from structurally related compounds, methyl glyoxal and acrolein, because it produces frameshift mutations whereas the latter compounds induce base-pair substitutions. MDA forms several adducts with deoxyguanosine but it is not certain which one causes the frameshift mutations. Ideally, a single adduct on a replicating genome could simulate the in vivo process by which MDA mutagenesis would occur. This technique is known as site-specific mutagenesis. The approach of choice for site-specific mutagenesis experiment is the gapped heteroduplex method which requires the formation of a small oligonucleotide with an adduct at a defined site. The construction of the oligonucleotide with the site specific adduct was attempted by ligating an adducted bisphosphate to the oligonucleotide, dCpdGpdC, with T_4-RNA ligase. The synthesis of 3’, 5’-bisphosphoryl-7-methyl-3-(β3-D-pentofuranosyl)-pyrimido [1, 2-a] purine-10-(3H)-one-2’-deoxyribose (7-Me-PyP-pdRp) was attempted. Although modification of the synthesis of 7-methyl- 3-(β-D-pentofuranosyl)-pyrimido [1, 2-a] purine-10-(3H)-one-2’-deoxyribose, the precusor to 7-Me-PyP-pdRp, with MgSO_4 and minimal concentrations of reactants improved the yield to 25%, the bisphosphate could not be formed. An adduct similar in structure to the MDA adduct was employed. The adduct, formed from 2, 3-epoxybutanalunder basic conditions in a 35% yield, was 3’, 5’-bisphosphoryl-l, N_2-etheno-2’-deoxyguanosine.
Substrates, pdεGp and dCpdGpdC, for the T_4-RNA ligase were successfully prepared. Ligation appeared successful, based on UV profiles, except possitive identification of the oligonucleotide product, dCpdGpdCpdεGp, was not possible due to low yields. Any modifications to increase the yield such as scale up attempts and polyethylene glycol addition were ineffective.
Carolin, Kathryn A., "Synthesis of site specific 1, n^2-ethenodeoxyguanosine in a small oligonucleotide" (1989). Wayne State University Theses. 606.