The mutagenic, carcinogenic and antineoplastic activities of N- nitroso compounds are related to their ability to covalently modify DNA. The structure and relative yields of adducts have been well characterized, but information concerning the reaction mechanism, sequence specificity and significance of DNA alkylation is still not fully understood. We have previously prepared N-alkyl-N- nitrosoureas (ANU, A=CH3 (MNU), CH2CH3 (ENU), CH2CH2Cl (Cl-ENU)), that are linked to a non-specific intercalating methidium and A-T minor groove binding lexitropsins (DNA reading N-methylpyrrole peptide). In contrast to MNU, the MNU-methidium compound generates N7-methylguanine (N7-MeG), as determined by polyacrylamide sequencing gels (PASG), without any base specificity and it is a better methylating agent at physiological ionic strength (200 mM NaCl). The MNU-lexitropsin compounds are poor alkylating agents, but in contrast to MNU, their alkylating activity is enhanced in the presence of the monocationic lexitropsin, distamycin A. Finally, the Cl-ENU-lexitropsins selectively alkylate an A and a T on the 3'-end of its affinity binding site. The data suggest that we have been successful in designing molecules that can site and/or sequence specifically methylate DNA due to the DNA affinity binding portion of the molecule. It is proposed to: 1) determine the complete profile of CH3, CH2CH3 and CH2CH2Cl adducts (including potential crosslinks) formed from (3H)MNU-, (3H)ENU- and (3H- ethyl)Cl-ENU-methidium and lexitropsins; 2) to characterize the affinity binding properties (binding geometry, sequence preference and association constants) for ANU-methidium and ANU-lexitropsin, and/or their denitrosated analogues; 3) to simultaneously compare the formation of all G-adducts from MNU. MNU-methidium and MNU- lexitropsin at oligo d(G)4 and d(G)I sequences; 4) to explore by PASG the mechanism responsible for the unique N7-MeG profile observed in oligo d(G) runs using synthetic oligomers containing two separated G4 runs, with one being made of non-ionic methylphosphonate linkages; 5) to determine if 5-methylcytosine residues generated by restriction methylation or major groove glycosylated cytosines in T4 bacteriophage DNA will effect DNA adduction by MNU, MNU-methidium and MNU-lexitropsin; and 6) to synthesize and characterize the DNA adduction by O-alkyl (alkyl=CH3, CH2CH3 and CH2CH2Cl) sulfonate esters that are covalently tethered to methidium and lexitropsins. The goal of the proposed research is to learn how to control covalent modification of DNA to better understand the effect of DNA adduction and to prepare more effective antineoplastic agents.
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