Abstract 1258 Synthesis of Oligonucleotides Containing DNA Adducts from a Novel Mitomycin C Derivative

Mitomycin C is a common anti-tumor drug currently used to treat breast, stomach and pancreatic cancers. This drug works by forming interstrand crosslinks (ICLs) between guanosine within opposing DNA strands (dG-ICL-dG), therefore, preventing DNA replication and transcription. DELS-15 is a derivative of mitomycin C which also forms ICLs with DNA in cells. Although the structure of MC and DELS-15 are similar, DELS-15 was found to be more cytotoxic toward tumor cells with a mutated TP53 gene. Since at least 50% of cancer cells lack a functioning TP53, understanding the molecular mechanism for the enhanced cytotoxicity of the novel MC derivative is crucial to develop more efficient therapeutics against tumor cells with a mutated TP53. Our hypothesis is that the different structures of MC and DELS-15 ICLs are responsible for the distinct cytotoxicity of the 2 drugs. In support of our hypothesis, we performed molecular modelling which showed that DNA distortion induced by DELS 15-ICL is much greater than in the case of MC-ICL. Our goal is to synthesize oligonucleotides bearing a single DELS-15 ICL at a specific site and, using these substrates, to decipher the molecular mechanisms responsible for the increased cytotoxicity of DELS-15 ICLs versus MC-ICLs. We developed a new biomimetic protocol to generate DELS-15 monoadducted oligonucleotides; the first step toward the synthesis of DELS-15 ICLs. The monoalkylated oligonucleotides were isolated by HPLC and the site of alkylation as well as the structure of the adducted oligonucleotides was confirmed using enzymatic digestion, UV, and LC-MS. We observed that this method also generated deoxyadenosine (dA) monoadducts but that formation of these dA adducts could be prevented by carefully selecting the sequence of alkylation. We then generated DELS-15 ICL by annealing a monoadducted 25-mer with a complementary strand and further reducing the monoadduct with sodium dithionite. In conclusion, we successfully developed a new protocol to access oligonucleotides containing DNA monoadducts and ICLs formed in cells by a novel Mitomycin C Derivative. These oligonucleotides will be used to delineate molecular pathways triggered by DNA ICLs inducing large DNA distortion and responsible for the enhanced cytotoxicity of DELS-15 toward cancer cells lacking a functioning TP53 gene. This research was supported by a grant from the National Institute of Health (1R16GM149514-01)to E.C.