Chemistry & Biochemistry Theses and Dissertations
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Item Target Alkylation of Single and Double Strand DNA by Peptide Nucleic Acids(2011) Liu, Yang; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quinone methides (QMs) generated in vivo can alkylate DNA and function as anti-cancer drugs. Delivery of QMs to target DNA is necessary to reduce the side effects caused by indiscriminate reaction. Previous, DNA was conjugated with a QM and was successfully used to deliver this QM to complementary DNA sequences. Peptide nucleic acids (PNAs) conjugates of QM are now being developed for in vivo application since PNA binds to its complementary DNA or RNA and PNA resists degradation by nucleases and proteases. The PNA1-QMP1 conjugate is capable of alkylating more than 60% of a complementary ssDNA when added at nearly stoichiometric quantities. No alkylation was observed if non-complementary DNA was treated with the conjugate. PNA1-QMP1 can alkylate a non-complementary DNA only when both the PNA and DNA target bind to a template strand. When no target sequences were present in solution, QM can react with nucleophiles from PNA1 and generate PNA1-QM1 self adduct. ssDNA can be alkylated by PNA1-QM1 self adduct with a 40% yield. The self adduct can survive after an incubation for 7 days in aqueous solution and preserve half of its original ability to alkylate complementary DNA. The reversibility and stability of the self adduct suggest that it can be used in cells. ssRNA can also be recognized and modified by PNA conjugates with a similar yield as earlier demonstrated with ssDNA. A PNA1-QM1 self adduct may also function as a telomerase inhibitor by alkylating RNA within telomerase. Polypyrimidine PNAs were prepared to bind to the major groove of duplex DNA selectively and expand the potential targets from single to double strand DNA. A cytosine-rich PNA recognized dsDNA and delivered an electron-rich QMP2 to its target sequences. The polypurine strand within a target dsDNA was alkylated at 37°C with a yield of 26%. PAN-QMP2 also showed strong selectivity toward its fully matched dsDNA over one base mismatch in the triplex recognition site. Successful delivery of a QMP to target single and double strand DNA by PNAs confirms that the use of PNA in vivo to target pre-selected sequences is feasible.Item REVERSIBLE QUINONE METHIDE ALKYLATION OF DNA(2009) Wang, Huan; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alkylation of DNA has been found to cause cancer and also to serve as its treatment. Quinone methides (QMs) are highly electrophilic molecules implicated in numerous metabolism processes. Studies of QM's reversible reaction with nucleophiles of DNA are important to understand the mechanism of its biological activity. Reversible alkylation of QMs can extend their lifetime under aqueous conditions. The repeated capture and release of QM from dA adduct can help QM equivalents escape the irreversible trapping and extend QM's lifetime by 100-fold. This effect of dA saturates at a concentration of about 6 mM. In contrast, dG, dC, and dT do not have the ability to preserve QM under aqueous conditions. Oligonucleotides can also preserve QM equivalents by forming labile intrastrand adducts. An oligonucleotide has now been shown to transfer bisQM to its complementary sequences to form interstrand crosslinking. Non-complementary sequences can not be alkylated by bisQM-oligonucleotide adducts. The nucleotide composition of oligonucleotides affects their ability to transfer QM as well. A G rich sequence showed a strong ability for crosslinking a complementary sequence. However, C rich and A rich sequences did not have such an ability. Excess alkylation of C rich and A rich oligonucleotides relative to that of G rich oligonucleotide may interrupt the hybridization of complementary sequences and suppress the formation of DNA crosslinking. The reversibility of crosslinking by QM within duplex DNA has been demonstrated by a strand displacement system. The reversible QM-DNA bond does not prevent strand displacement and allows bisQM to migrate within a series of changing DNA structures by forming crosslinking. The reactivity of bisQM is preserved beyond 11 days in duplex DNA by forming labile DNA cross-links under aqueous conditions. The migration of QM is found to be under thermodynamic control and bisQM preferentially retain cross-links in the most stable DNA duplexes.Item Selective Delivery of a Quinone Methide Precursor by Peptide Nucleic Acids(2007-12-10) Liu, Yang; Rokita, Steve; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quinone methides (QMs) are electrophilic intermediates that can be generated in vivo to alkylate DNA and function as anti-cancer drugs. Previously, DNA-QM conjugates have shown the ability to selectively deliver a QM to specific sequences of DNA. Peptide nucleic acids (PNAs) conjugates of QM are now being developed since PNA binds DNA with higher affinity than natural DNA. Synthesis of PNA oligomers and conjugation of the PNA to QM precursor are reported here. Synthesis of peptides was used to study the optimum conditions for preparation of the ultimate peptide-PNA conjugate. Both peptides and peptide-PNA have been synthesized after optimizing solid-phase techniques. Conditions for coupling a quinone methide precursor (QMP) and peptide-PNA conjugates were also evaluated. 8-Amino-3,6-dioxaoctanoic acid that links PNA and QMP is essential for coupling. MOPS buffer containing the peptide-PNA and an acetonitrile/dimethylformamide mixture containing QMP were combined for coupling. Finally, reactive QM derivative of peptide-PNA-QM was studied.