Biphotonic photoionization of frozen aqueous solutions of DNA at 248 nm has been shown by EPR spectroscopy to lead selectively to the guanine cation. In H 2 18 O under these conditions high levels of [ 18 O]-7,8dihydro-8-oxo-2′-deoxyguanosine are produced in a dose-dependent manner, confirming direct formation of this oxidation product by hydration of the guanine cation. Photoionization of defined oligonucleotides did not give rise to significant levels of immediate strand breaks but generated G-specific alkali-labile sites that are readily cleaved by piperidine treatment. Authentic oligonucleotides containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG, 5) sites are slowly cleaved at these sites on treatment with piperidine but at rates inconsistent with this being the source of the alkali-labile site. Photoionization of 7,8-dihydro-8-oxo-2′-deoxyguanosine-containing oligonucleotides demonstrated that this residue is highly susceptible to secondary oxidation and leads to formation of a markedly more alkali-labile lesion. Photoionization of 7,8-dihydro-8-oxo-2′-deoxyguanosine leads to release of 7,8-dihydro-8-oxoguanine suggesting that the alkali-labile site on photoionization of 7,8-dihydro-8-oxo-2′-deoxyguanosinecontaining oligonucleotides is an apurinic site. However, the lack of significant 7,8-dihydro-8-oxoguanine release on photoionization of the parent oligonucleotides rules out secondary oxidation of 7,8-dihydro-8-oxo-2′-deoxyguanosine as the mechanism of formation of alkali-labile sites.

Singlet oxygen quantum yields from the triplet excited states of DNA, nucleotides, dinucleosides, purine and pyrimidine bases in solution have been determined-for guanine-containing moieties no singlet oxygen was detected, and possible implications pertaining to DNA photodamage are discussed.

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The mechanism and kinetics of hydrolysis of chlorambucil and chlorambucil-spennidine conjugate in aqueous buffered solutions have been compared. In the absence of added chloride ion the reactions are shown to be first-order in the nitrogen mustard and independent of the nucleophile concentration. In the presence of high concentrations of sodium chloride the reaction is reversible and is subject to a significant common-ion effect. The rates of hydrolysis of both compounds are independent of pH in the range 8 to 3.5, and both rates begin to drop rapidly below pH 3.5 which corresponds to the pK,s of the aryl amine groups. The relative rates of alkylation of a range of nucleophiles by chlorambucil have been deduced from the isokinetic points, and have shown that the phosphate dianion, imidazole base and particularly thiolates are all capable of competing with water for the aziridinium ion at comparatively low concentrations. The rates of reaction of chlorambucil and the chlorambucil-spermidine conjugate have been shown to be sensitive to the medium, and, in particular, there is a large micellar inhibition of the hydrolysis of chlorambucil(60-fold reduction in rate) in the presence of hexadecyltrimethylammonium chloride that is not seen for the conjugate. These data are all accounted for in terms of a rate limiting formation of the aziridinium ion intermediate in each case. No evidence for any other mechanistic pathways was found.

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