Abstract

Cellular DNA is susceptible to damage by chemical agents that causes modifications which includes interstrand cross-links (ICL). ICLs covalently attach the complementary DNA strands, and interfere with replication and transcription by preventing strand separation. The deliberate formation of ICL in DNA by bi-functional alkylating chemotherapeutic agents leads to the death of cancer cells. Development of tumors resistant to these agents is a factor in the lack of response in some patients, with removal of ICL believed to play a role in resistance. In mammalian cells, the precise role of excision repair in eliminating ICL is not completely understood. For better understanding of the repair pathways involved in removing ICL damage, ICL DNA duplexes containing well-defined modified moieties are required to mimic the lesions induced by chemotherapeutic agents.
This thesis describes two major approaches that have been investigated to synthesize ICL DNA. The first describes a method to prepare DNA duplexes containing cross-linked N3-butylene-N3 thymidines that enables the preparation of asymmetric nucleotide sequences around cross-linked sites. Protective groups for the 3’- and 5’-hydroxyl moieties were screened for compatibility of subsequent extensions from a cross-linked thymidine dimer incorporated in a support bound oligonucleotide by automated DNA synthesis. Two cross-linked dimer phosphoramidites were prepared, one with dimethoxytrityl (DMT) and allyloxycarbonyl (Alloc) protective groups at 5’-O positions and a 3’-O-t-butyldimethylsilyl (TBS) group which enabled the production of completely asymmetric ICL DNA duplexes in good yields. After coupling of the cross-linked phosphoramidite to a linear strand assembled on the solid support, the DMT group was cleaved on the synthesizer to allow for the synthesis of the second arm of the duplex. The Alloc group was then removed via an off-column strategy to expose the 5'-hydroxyl group to complete assembly of one strand of the duplex to form a "Y-shaped" intermediate. Final removal of a 3'-O-TBS group off column followed by coupling with deoxynucleoside 5'-phosphoramidites yielded ICL DNA duplexes containing completely asymmetric nucleotide composition around the cross-link site. The identity and composition of the ICL duplexes were confirmed by mass spectrometry (ESI-TOF) and enzymatic digestion. The synthesized ICL duplexes displayed characteristic features of a B-form duplex and had stabilities that were higher than those of the unmodified controls assessed by circular dichroism (CD) spectroscopy and UV thermal denaturation experiments.
The second major project describes approaches to prepare a 7-deaza-2’-deoxyguanosine cross-linked dimer where the C7 atoms are attached by an alkylene linker. The chemical instability of alkylated N7 2’-deoxyguanosine (dG) represents a major challenge for preparing ICL DNA containing an alkylene linkage between the N7 atoms. The incorporation of a C7-alkylene cross-linked dimer of 7-deaza-2’-deoxyguanosine in DNA would allow for the preparation of a chemically stable ICL which mimic lesions formed by bifunctional alkylating agents (i.e. mechlorethamine and hepsulfam). Two synthetic methods were explored to prepare 7-deazaguanine (and other 7-deazapurines). These involved two cyclization strategies to prepare these molecules starting from a pyrimidine or a pyrrole to produce the purine. In both synthetic methods it was challenging to purify some of the intermediates. All intermediates in the synthetic method starting from the pyrimidine precursors to produce the 7-deazapurines were more stable while the production of the 7-deazapuines from the pyrroles resulted in higher yields.
An attempt to produce a C7 cross-linked dimer of 7-deaza-2’-deoxyguanosine containing a heptamethylene linker is described. Starting from 7-deazaguanine, 7-iodo-7-deaza-2’-deoxyguanosine was prepared in good yield. This nucleoside was converted to 5’-O-DMT-7-iodo-7-deaza-2’-deoxyguanosine and the Sonogashira reaction used with 1,6-heptadiyne to introduce the heptamethylene linker. Unfortunately, multiple challenges were encountered with the dimerization and hydrogenation reactions which did not allow for the synthesis of the desired dimer for solid-phase synthesis of the ICL DNA.