0(-Methylguanine (m6G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3' side of the modified base. Templates were primed with oligonucleotides terminating one or two bases prior to the site at which incorporation kinetics were to be investigated. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent Km and relative V,,x of incorporation of either dCTP or dTTP opposite m6G or G.These data were used to calculate the relative frequency of incorporation opposite the m6G or the unmodified G. When the sequence was 3'-Cm6G-5', there was a 6-to 7-fold preference for formation of a m6G-T pair compared with m6G-C. The m6G-T frequency, based on Vmix/Km, was at least 50-fold greater than that of a G-T pair at the same site. Changing the sequence to 3'-Tm6G-5' had a marked effect on both Km and V.,, of pairs containing m6G and on the incorporation frequency of T opposite m6G, which was then only slightly favored over m6G-C. When replication was started directly opposite m6G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m6G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3' position can have a major effect on mutagenic efficiency. Replication using Drosophila Pol a gave the same values for relative frequencies. Pairing of either C or T with m'G on the primer terminus did not significantly inhibit extension of the next normal base pair, in contrast to terminal mismatches of unmodified bases. It is concluded that, in the absence of repair, m6G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated base. This variation in frequency of changed pairing could contribute to the occurrence of mutational "hot spots" after replication of damaged DNA.The likely role of 06-alkylguanines as a major factor in mutagenesis by certain alkylating agents was recognized by Loveless about 20 years ago (1). In an attempt to resolve contradictions in the literature, he suggested that O6-methylguanine (m6G) might pair with thymine (T) during replication and thus cause the G-C -* A-T transitions found as the major genetic change after reaction with certain carcinogenic alkylating agents. Further studies in a number of laboratories established that the occurrence, and in particular the persistence of this alkylated base, often correlated with the biological endpoints of mutagenesis and carcinogenesis (2). In accord with the proposed mechanism of mutagenesis, m6G can pair with T both in vitro (3) and in vivo (4), when presented to a polymerase either in the template or as a precursor to DNA synthesis. This model of mutagenesis occurring by base pairing between m6G and T during replication has long been assumed, but only recently has it begun to be tested rigorously [reviewed by Basu and Essigmann (5)]. When an m6G-containing dodecamer was annealed with a series of anal...

Although Co(III)-alkyl peroxo species have often been implicated as intermediates in industrial oxidation of hydrocarbons with cobalt catalysts, examples of discrete [LCo III -OOR] complexes and studies on their oxidizing capacities have been scarce. In this work, twelve such complexes with two different ligands, L, and various primary, secondary, and tertiary R groups have been synthesized, and seven of them have been characterized by X-ray crystallography. The dianion (L 2-) of the two ligands N, N-bis[2-(2-pyridyl)ethyl]pyridine-2,6-dicarboxamide (Py 3 PH 2 , 1) and N, N-bis[2-(1-pyrazolyl)ethyl]pyridine-2,6-dicarboxamide (PyPz 2 -PH 2 , 2) bind Co(III) centers in pentadentate fashion with two deprotonated carboxamido nitrogens in addition to three pyridine or one pyridine and two pyrazole nitrogens to afford complexes of the type [LCo III (H 2 O)] and [LCo III (OH)]. Reactions of the [LCo III (OH)] complexes with ROOH in aprotic solvents of low polarity readily afford the [LCo III -OOR] complexes in high yields. This report includes syntheses of [Co(Py 3and [Co(PyPz 2 P)(OOR)] complexes with R ) t Bu (13), Cm (14), CMe 2 CH 2 Ph (15), Cy (16), i Pr (17) or n Pr (18). The structures of 8-12 and 16 have been established by X-ray crystallography. Complexes 10 and 16 are the first examples of structurally characterized compounds containing the [Co-OOCy] unit, proposed as a key intermediate in cobalt-catalyzed oxidation of cyclohexane. The metric parameters of 7-12 and 16 have been compared with those of other reported [LCo III -OOR] complexes. When these [LCo III -OOR] complexes are warmed (60-80 °C) in dichloromethane in the presence of cyclohexane (CyH), cyclohexanol (CyOH) and cyclohexanone (CyO) are obtained in good yields. Studies on such reactions (referred to as stoichiometric oxidations) indicate that homolysis of the O-O bond in the [LCo III -OOR] complexes generates RO • radicals in the reaction mixtures which are the actual agents for alkane oxidation. [LCo-O• ], the other product of homolysis, does not promote any oxidation. A mechanism for alkane oxidation by [LCo III -OOR] complexes has been proposed on the basis of the kinetic isotope effect (KIE) value (5 at 80 °C), the requirement of dioxygen for oxidation, the dependence of yields on the stability of the RO • radicals, and the distribution of products with different substrates. Both L and R modulate the capacity for alkane oxidation of the [LCo III -OOR] complexes. The extent of oxidation is noticeably higher in solvents of low polarity, while the presence of water invariably lowers the yields of the oxidized products. Since [LCo III -OOR] complexes are converted into the [LCo III (OH)] complexes at the end of single turnover in stoichiometric oxidation reactions, it is possible to convert these systems into catalytic ones by the addition of excess ROOH to the reaction mixtures. The catalytic oxidation reactions proceed at respectable speed at moderate temperatures and involve [LCo III -OOR] species as a key intermediate. Turnover numbers over 1...

Three cobalt(III) complexes of Py3PH2 (H's are the dissociable amide H's), a strong-field ligand with two peptide groups, have been synthesized. They are [Co(Py3P)(H2O)]ClO4·H2O (6), [Co(Py3P)(OH)] (7), and [Co(Py3P)(OO t Bu)]·2CH2Cl2 (8). Complex 6 crystallizes in the monoclinic space group C2 with a = 22.695(6) Å, b = 10.284(2) Å, c = 10.908(3) Å, α = 90°, β = 112.17(2)°, γ = 90°, V = 2357.7(10) Å3, and Z = 4. Its structure has been refined to R = 3.91% on the basis of 1844 I > 2σ(I) data. Complex 8 crystallizes in the monoclinic space group P21/n with a = 16.720(4) Å, b = 10.641(3) Å, c = 16.776(3) Å, α = 90°, β = 99.76(2)°, γ = 90°, V = 2941.5(12) Å3, and Z = 4. The structure of 8 has been refined to R = 6.37% on the basis of 4776 I > 2σ(I) reflections. In all three complexes, the doubly deprotonated Py3P2- ligand binds the cobalt(III) center in a pentadentate fashion with five nitrogens situated in two deprotonated amido groups and three pyridine rings. The aqua complex 6 can easily be converted into the hydroxo complex 7 by the addition of 1 equiv of base. The transformation 6 ↔ 7 is reversible, and the pK a of the coordinated water molecule in 6 is 7. Complex 8 is the first example of a structurally characterized Co(III) alkyl peroxide complex that contains two deprotonated amido groups bonded to the metal center. Like a few alkyl peroxide complexes of tervalent cobalt, 8 oxidizes alkanes upon thermal decomposition. When cyclohexane is used as the substrate, cyclohexanol, cyclohexanone, and cyclohexyl chloride are the products. Complex 7 is the intermediate in the formation of 8 and is also the thermal decomposition product of 8. In single turnover oxidation of cyclohexane by 8 at the optimum temperature of 80 °C, a maximum yield of 59% of the oxidized products is obtained. The mechanism of cyclohexane oxidation by 8 involves exclusive homolytic scission of the O−O bond in 8. The t BuO• radicals generated in such a process abstract an H atom from cyclohexane to afford cyclohexyl radicals, which in turn react with dioxygen and produce cyclohexanol and cyclohexanone presumably via a Russell-type termination reaction. The oxidation of cyclohexane by 8 can be either stoichiometric or catalytic. In the presence of excess TBHP, 8 affords more oxidized products, indicating multiple turnovers.