Date of Completion

8-22-2013

Embargo Period

8-22-2013

Keywords

DNA Damage, Cyclo-dG, N-Glycosidic Bond Stability, Carcinogen, Nucleosides, Triphosphate

Major Advisor

Dr. Ashis K. Basu

Associate Advisor

Dr. Steven L. Suib

Associate Advisor

Dr. Xudong Yao

Field of Study

Chemistry

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

8,5’-Cyclopurine deoxynucleosides are unique tandem lesions containing an additional covalent bond between the base and the sugar. These mutagenic and genotoxic lesions are repaired only by nucleotide excision repair (NER). It was shown that the R-cdA is repaired more efficiently than the S-cdA by NER. The research on cyclopurines has been focused on 8,5’-cyclo-2’-deoxyadenosine (cdA) and its riboside analog, whereas relatively little is known about 8,5’-cyclo-2’-deoxyguanosine (cdG), except a preliminary study reported the latter to be a strong block of replication in vitro.

To study the structural and biological effects of these DNA lesions, I have used the total synthesis approach to incorporate the R and S-cdG lesions into oligonucleotides. The key step of formation of the 8,5’ bond in dG was carried out by radical cyclization using thiophenyl group as a photolabile precursor of the 5’-C radical. SeO2 oxidation of C-5’ followed by sodium borohydride reduction gave S-cdG. R-cdG was synthesized by inversion of 5’-OH of S-cdG. The protected monomers of these DNA lesions were prepared by well-established methods, and oligonucleotides were synthesized by standard techniques. In collaborative work, the solution structures of DNA duplexes containing a site-specific S-cdG lesion placed opposite dC, dT, or dA in the complementary strand were obtained by molecular dynamics calculations restrained by distance and dihedral angle restraints attained from NMR spectroscopy. Biological studies have shown that S-cdG induces DNA polymerase V-dependent mutations at a frequency of 34% in Escherichia coli. Most are S-cdG → A transitions, suggesting misincorporation of dTMP occurred during replication bypass of this lesion.

The N-glycosidic (or C1’-N9) bond of 2’-deoxyguanosine (dG) derivatives is usually susceptible to acid hydrolysis, but even after cleavage of this bond of the cyclopurine lesions, the base would remain attached to the sugar. Here, the stability of the N-glycosidic bond and the products formed by formic acid hydrolysis of (5’S)-8,5’-cyclo-2’-deoxyguanosine (S-cdG) were investigated. For comparison, the stability of the N-glycosidic bond of 8,5’-cyclo-2’,5’-dideoxyguanosine (ddcdG), 8-methyl-2’-deoxyguanosine (8-Me-dG), 7,8-dihydro-8-oxo-2’-deoxyguanosine (8-Oxo-dG), and dG was also studied. In various acid conditions, S-cdG and ddcdG exhibited similar stability to hydrolysis. Likewise, 8-Me-dG and dG showed comparable stability, but the half lives of the cyclic dG lesions were 5-10-fold higher than dG or 8-Me-dG. NMR studies were carried out to investigate the products formed after the cleavage of the C1’-N9 bond. 2-Deoxyribose generated α and β anomers of deoxyribopyranose and deoxyribopyranose oligomers following acid treatment. S-cdG gave α and β deoxyribopyranose linked guanine as the major products, but α and β anomers of deoxyribofuranose linked guanine and other products were also detected.

In order to facilitate NMR studies and low level detection in biological samples, [1,3, NH2-15N3] (5’S)-8,5’-cyclo-2’-deoxyguanosine was synthesized from imidazole-4,5-dicarboxylic acid in twenty-one steps. The three 15N isotopes were introduced during the chemo-enzymatic preparation of [1,3, NH2-15N3]-2’-deoxyguanosine using an established procedure. The [1,3, NH2-15N3]-S-cdG was prepared similarly like ­S-cdG. The isotopic purity of the [1,3, NH2-15N3] (5’S)-8,5’-cyclo-2’-deoxyguanosine was in excess of 99.94 atom% based on LC-MS measurements and was suitable for using as a biomarker.

Nucleoside analogues are widely used as antiviral agents for treatment of viral diseases. Most of these drugs function through the 5’-triphosphate as the active metabolite. I have synthesized (5’S)-8,5’-cyclo-2’-deoxyguanosine triphosphate (cdGTP) for studying the effects of incorporation into DNA. Synthesis of S-cdGTP synthesis has been carried out using 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one as an active phosphitylating agent. The reaction was monitored using 31P NMR. The synthesized triphosphate is of high quality and suitable for incorporation into DNA.

6-Nitrochrysene (6-NC) is a nitro polynuclear aromatic hydrocarbon (PAH) found in many high temperature exhaust mixtures like coke oven exhaust or consumed engine oil. 6-NC is the most active and potent tumorigen in experimental animals among all tested nitroaromatic compounds. Also, the carcinogenic potency of 6-NC is one of the highest amongst many different metabolites of PAHs. I have synthesized the protected phosphoramidite of the C8 2’-deoxyguanosine adduct of 6-aminochrysene by Buchwald-Hartwig Pd-catalyzed coupling. This phosphoramidite will be used for incorporation into oligonucleotides for structural and biological studies.

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