A series of 7-aryl- and 7-hetaryl-7-deazaadenosines was prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.

Modified dATP (2'-deoxyadenosine-5'-triphosphate) and dUTP (2'-deoxyuridine-5'-triphosphate) bearing ferrocene (Fc) labels linked via a conjugate acetylene spacer were prepared by single-step aqueous-phase cross-coupling reactions of 7-iodo-7-deaza-dATP or 5-iodo-dUTP with ethynylferrocene. The Fc-labeled dNTPs were good substrates for DNA polymerases and were efficiently incorporated to DNA by primer extension (PEX). Electrochemical analysis of the 2'-deoxyribonucleoside triphosphates (dNTPs) and PEX products revealed significant differences in redox potentials of the Fc label bound either to U or to 7-deazaA and between isolated dNTPs and conjugates incorporated to DNA. Prospective bioanalytical applications are outlined.

DNA polymerases accurately replicate DNA by incorporating mostly correct dNTPs opposite any given template base. We have identified the chemical features of purine dNTPs that human pol α uses to discriminate between right and wrong dNTPs. Removing N-3 from guanine and adenine, two high fidelity bases, significantly lowers fidelity. Analogously, adding the equivalent of N-3 to lowfidelity benzimidazole-derived bases (i.e., bases that pol α rapidly incorporates opposite all 4 natural bases) and to generate 1-deazapurines significantly increases the ability of pol α to identify the resulting 1-deazapurines as wrong. Adding the equivalent of the purine N-1 to benzimidazole or to 1-deazapurines significantly decreases the rate at which pol α polymerizes the resulting bases opposite A, C, and G, while simultaneously enhancing polymerization opposite T. Conversely, adding the equivalent of adenine's C-6 exocyclic amine (N-6) to 1-and 3-deazapurines also enhances polymerization opposite T, but does not significantly decrease polymerization opposite A, C, and G. Importantly, if the newly inserted bases lack N-1 and N-6, pol α does not efficiently polymerize the next correct dNTP, whereas if it lacks N-3 one additional nucleotide is added and then chain termination ensues. These data indicate that pol α uses two orthogonal screens to maximize its fidelity. During dNTP polymerization, it uses a combination of negative (N-1 and N-3) and positive (N-1 and † This work was supported by grants to RDK from the NIH (GM54194 and TW007372-01) and the Army Research Office (W911NF-05-1-0172), to MH from the Ministry of Education of the Czech Republic (Centre of Biomolecules and Complex Molecular Systems, LC 512), and to JE from the Deutsche Forschungsgemeinschaft (SFB 579).