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Nucleoside configuration (α-d vs. β-d), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2'-deoxyguanosines and anomeric 2'-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2'-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d/α-d and β-d/β-d homo base pairs or α-d/β-d and β-d/α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective Tm stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d/β-d or α-d/α-d nucleoside combinations are more stable than α-d/β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.

Novel silver-mediated dA-dC, dA*-dC, and dA*-dG base pairs were formed in a natural DNA double helix environment (dA* denotes 7-deaza-dA, 7-deaza-7-iodo-dA, and 7-cyclopropyl-7-deaza-dA). 7-Deazapurine nucleosides enforce silver ion binding and direct metal-mediated base pair formation to their Watson-Crick face. New phosphoramidites were prepared from 7-deaza-dA, 7-deaza-7-iodo-dA, and 7-cyclopropyl-7-deaza-dA, which contain labile isobutyryl protecting groups. Solid-phase synthesis furnished oligonucleotides that contain mismatches in near central positions. Increased thermal stabilities (higher Tm values) were observed for oligonucleotide duplexes with non-canonical dA*-dC and dA-dC pairs in the presence of silver ions. The stability of the silver-mediated base pairs was pH dependent. Silver ion binding was not observed for the dA-dG mismatch but took place when mismatches were formed between 7-deazaadenine and guanine. The specific binding of silver ions was confirmed by stoichiometric UV titration experiments, which proved that one silver ion is captured by one mismatch. The stability increase of canonical DNA mismatches might have an impact on cellular DNA repair.

One-electron photooxidations of 5-methyl-2'-deoxycytidine (d(m)C) and 5-trideuteriomethyl-2'-deoxycytidine ([D(3)]d(m)C) by sensitization with anthraquinone (AQ) derivatives were investigated. Photoirradiation of an aerated aqueous solution containing d(m)C and anthraquinone 2-sulfonate (AQS) afforded 5-formyl-2'-deoxycytidine (d(f)C) and 5-hydroxymethyl-2'-deoxycytidine (d(hm)C) in good yield through an initial one-electron oxidation process. The deuterium isotope effect on the AQS-sensitized photooxidation of d(m)C suggests that the rate-determining step in the photosensitized oxidation of d(m)C involves internal transfer of the C5-hydrogen atom of a d(m)C-tetroxide intermediate to produce d(f)C and d(hm)C. In the case of a 5-methylcytosine ((m)C)-containing duplex DNA with an AQ chromophore that is incorporated into the backbone of the DNA strand so as to be immobilized at a specific position, (m)C underwent efficient direct one-electron oxidation by the photoexcited AQ, which resulted in an exclusive DNA strand cleavage at the target (m)C site upon hot piperidine treatment. In accordance with the suppression of the strand cleavage at 5-trideuterio-methylcytosine observed in a similar AQ photosensitization, it is suggested that deprotonation at the C5-methyl group of an intermediate (m)C radical cation may occur as a key elementary reaction in the photooxidative strand cleavage at the (m)C site. Incorporation of an AQ sensitizer into the interior of a strand of the duplex enhanced the one-electron photooxidation of (m)C, presumably because of an increased intersystem crossing efficiency that may lead to efficient piperidine-induced strand cleavage at an (m)C site in a DNA duplex.

This paper focuses on the inhibition of ribonucleotide reductase (RNR) by gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), a deoxycytidine analogue that is a very active drug against solid tumors and is currently commercialized as gemzar. RNR inactivation is reductant-dependent and occurs in a very different way from that of other known substrate analogues. In the presence of reductants monomer R1 of RNR is inhibited, whereas in the absence of reductants the radical is lost and monomer R2 is inhibited. As inside the cell reductants are available, it is likely that R1 inactivation is the most favorable mechanism responsible for drug cytotoxicity. This inhibition pathway has been unknown to date, but we have conducted a theoretical study that has led us to the first proposal of a mechanism for RNR inhibition by dFdC in the presence of reductants.

The four non‐canonical nucleotides in the human genome 5‐methyl‐, 5‐hydroxymethyl‐, 5‐formyl‐ and 5‐carboxydeoxycytidine (mdC, hmdC, fdC and cadC) form a second layer of epigenetic information that contributes to the regulation of gene expression. Formation of the oxidized nucleotides hmdC, fdC and cadC requires oxidation of mdC by ten‐eleven translocation (Tet) enzymes that require oxygen, Fe(II) and α‐ketoglutarate as cosubstrates. Although these oxidized forms of mdC are widespread in mammalian genomes, experimental evidence for their presence in fungi and plants is ambiguous. This vagueness is caused by the fact that these oxidized mdC derivatives are also formed as oxidative lesions, resulting in unclear basal levels that are likely to have no epigenetic function. Here, we report the xdC levels in the fungus Amanita muscaria in comparison to murine embryonic stem cells (mESCs), HEK cells and induced pluripotent stem cells (iPSCs), to obtain information about the basal levels of hmdC, fdC and cadC as DNA lesions in the genome., Exact UHPLC‐MS2 quantifications were used to determine the oxidative lesion and epigenetically induced levels of the non‐canonical bases hmdC, fdC and cadC and of the levels of base excision repair intermediates.