Date of Completion

1-7-2019

Embargo Period

1-6-2023

Keywords

DNA damage, Polycyclic aromatic hydrocarbon, 3-nitrobenzanthrone, 6-aminochrysene, Alpha-amino-carboline, TLS polymerases

Major Advisor

Dr. Ashis K. Basu

Associate Advisor

Dr. Mark W. Peczuh

Associate Advisor

Dr. Christian Bruckner

Field of Study

Chemistry

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

A large number of nitro polycyclic aromatic hydrocarbons (nitro-PAHs) and heterocyclic aromatic amines (HAAs) are present in the environment and many compounds of these families are carcinogenic. 2ʹ-Deoxyribonucleic acid (DNA) adduct or lesion formation by these class of compounds play an important role in the process of carcinogenesis. Upon metabolic activation by cytochrome P450 family of enzymes, these nitro-PAHs or HAAs form electrophilic metabolites, which then reacts with the nucleic acid bases of DNA to form DNA-carcinogen adducts. Subsequent to DNA damage, activation of DNA damage checkpoints helps to arrest the growth of the damaged cell and allows the DNA repair mechanism to rectify the error. However, sometimes prior to DNA repair, cells can undergo replications, but replicative DNA polymerases, in most cases, cannot bypass a DNA damage. Depending on the nature of the damage, a special group of DNA polymerases known as translesion synthesis (TLS) DNA polymerases help to bypass this bulky adducts. But this damaged DNA replication by TLS polymerases can lead to mutations, which, in turn, may lead to production of altered proteins. If the mutation occurs in a tumor suppressor gene or oncogene, it can lead to human cancers.

3-Nitrobenzanthrone (3-nitro-7H-benz-[d,e]anthracen-7-one, 3-NBA), a member of the nitro polycyclic aromatic hydrocarbon (PAH), is found in exhausts from diesel and gasoline engines owing to incomplete combustion of the fuel and is considered an environmental pollutant and a suspected human carcinogen. 3-NBA is much more mutagenic than the well-studied PAH benzo[a]pyrene (B[a]P) and the most abundant nitroarene found in diesel exhaust, 1-nitropyrene (1-NP). It causes DNA damage and exhibits mutagenicity in bacterial and mammalian cells. 3-NBA is one of the most potent mutagens in the Ames’ assay. Upon metabolic activation, it forms N-(deoxyguanosin-8-yl)-3-amino-benzanthrone (C8-dG-ABA), the major alkylation adduct at the C8-position of 2ʹ-deoxyguanosine (dG). To investigate its replication properties, I have used a total synthesis approach to synthesize the C8-dG-ABA adduct. The adduct was incorporated into a defined site of a dodecamer oligodeoxynucleotide, 5’-CATG*ATGACGCT-3’, where G* represents the adducted dG and the bypass mechanism was analyzed using crystallographic techniques with the incoming 2ʹ-deoxycytosine triphosphate (dCTP).

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. Upon metabolic activation, 6-NC forms two major adducts with dG, one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of site-specific oligonucleotides containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC by Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative. The monomer for solid-phase DNA synthesis was prepared from this adduct by its deprotection followed by conversion to the corresponding phosphoramidite, which was used to synthesize the site-specific oligonucleotides 5’-GTGCG*TGTTTGT-3, which contains the local DNA sequence of the tumor suppressor gene p53 (codon 273 is underlined), where G* represents the C8-dG-6-Ac adduct. The adduct-containing oligonucleotides were purified by reversed phase HPLC followed by denaturing polyacrylamide gel electrophoresis and characterized by mass spectrometry. Oligonucleotide 5’-GTGCG*TGTTTGT-3 was then incorporated into a plasmid and replicated in human embryonic kidney 293T cells, which showed that the DNA adduct is mutagenic inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in its bypass using siRNA knockdown approach.

2-Amino-9H-pyrido [2, 3-b] indole (AαC) is the most abundant heterocyclic aromatic amine (HAA) and a possible liver and gastrointestinal carcinogen found in the mainstream tobacco smoke. Upon cytochrome P450 mediated N-oxidation of the exocyclic amine of AαC, it reacts to DNA to form N-(deoxyguanosin-8-yl)-AαC adduct (C8-dG-AαC). To study the structural and biological effects of C8-dG-AαC lesion, we conceived a total synthesis approach using 9-benzyl-9H- pyrido [2, 3-b] indol-2-amine and protected C8-bromo-2'-deoxyguanosine derivative as precursors to furnish the 2'-dG-C8-AαC adduct. The DNA adduct will be incorporated into defined sites of a dodecamer oligodeoxynucleotide, 5’-GTGCG*TGTTTGT-3’, as in the previous case to understand the carcinogenic potency and critical roles of human DNA polymerases in its translesion synthesis.

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