Mark E. Malone

Mechanism and reactivity of chlorambucil and chlorambucil–spermidine conjugate

The mechanism and kinetics of hydrolysis of chlorambucil and chlorambucil–spermidine 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 pKas 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.

Effects of ionizing radiation on deoxyribonucleic acid. Part 7. Electron capture at cytosine and thymine

Exposure of a range of DNA samples in various media to 60Co γ-rays at 77 K gives electron-capture centres characterized by an EPR doublet with A(1H) of ca. 16 G. Since the electron-adducts of C and T give very similar doublet EPR spectra in irradiated DNA, it is difficult to judge the proportions of C˙– and T˙– formation by inspection. The possibility, suggested by others, that computer fits can be used to give a quantitative measure of these species is discussed. However, in view of the variability of the features directly assignable to C˙– and T˙– units in different environments, we suggest that this approach has only qualitative significance.The alternative method involves annealing to convert T˙– into TH˙ radicals in which a hydrogen atom is added to C6, the resulting radical having a completely characteristic octet EPR spectrum. It is argued that the ejected electrons move through the stacked DNA bases, becoming trapped at C or T depending upon the relative rates at which C˙– and T˙– are protonated to give C˙–(H+)(protonated at N3) and T˙–(H+)(protonated on oxygen). If this is correct, interconversion between C˙– and T˙– on annealing is unlikely, and only T˙– can lead to TH˙ formation.This is also not accurate, since TH˙ decay sets in within the same temperature range as it is being formed. By generating TH˙ from frozen aqueous DNA with ultraviolet light, the pure decay annealing curve has been obtained, and using this we have been able to extrapolate the data for TH˙ from the γ-irradiated samples to give the real yields of TH˙. The results show that ca. 36% of the doublet must be due to T˙– centres, the remainder (64%) being assigned to C˙– centres.The ratio of C˙– to T˙– varies with the DNA source, and with the environment. We suggest that it is largely governed by the relative rates of protonation to give C˙–(H+) and T˙–(H+), and the factors controlling these rates are discussed. The use of lithium chloride glasses completely suppresses the formation of G˙+ centres, leaving well-defined radical-anion spectra, but on annealing, conversion to TH˙ is negligible despite the rapid, and complete, loss of the doublet species. This result is discussed in terms of reaction with Cl2˙– radicals formed in abundance in these glasses.Studies designed to detect any site-specificity in the DNA damage leading to strand breaks suggest that all possible sites are damaged. These results strongly support the postulate that yields of C˙– and T˙– are comparable. The possibility that some A˙+ cations are formed in addition to G˙+ cations is also considered in the light of these results.

Singlet oxygen sensitisation by excited state DNA

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 oxyge was detected, and possible implications pertaining to DNA photodamage are discussed.

Peroxidase Activation of 4-Hydroxytamoxifen to Free Radicals Detected by EPR Spectroscopy

4-Hydroxytamoxifen is a major metabolite of the antiestrogenic drug tamoxifen used in the treatment of women with breast cancer. 4-Hydroxytamoxifen is broken down by a horseradish peroxidase/H2O2 system very much more rapidly than tamoxifen and causes much greater DNA damage determined by 32P-postlabelling. EPR spin trapping of 4-hydroxytamoxifen reaction products in the presence of the free radical trap 5,5-dimethyl-1-pyrroline N-oxide, together with glutathione as a hydrogen donor, resulted in the generation of a species with the characteristics of the glutathione thiyl radical (aN approximately 15.3 G, aH approximately 16.2 G). Support for the creation of thiyl radicals comes from the close to stoichiometric time dependent formation of glutathione disulfide concomitant with the loss of glutathione. Similar results were obtained using 4-hydroxytoremifene but no radical formation or glutathione loss could be detected using 3-hydroxytamoxifen (droloxifene). On-line LC-ESI MS analysis of the incubation products from 4-hydroxytamoxifen has identified three products with a protonated molecular mass of 773, consistent with the formation of dimers of 4-hydroxytamoxifen. The role that radical mechanisms have in the carcinogenic effects of tamoxifen in the endometrium or other target organs of women taking this drug remains to be established.

Factors controlling the site of protonation of the one-electron adduct of cytosine and its derivatives

Exposure of cytosine and derivatives thereof to 60Co γ-rays at 77 K in a range of aqueous solutions (H2O or D2O) at 77 K gave EPR spectra assigned to the corresponding radical anions. These spectra comprise either doublet or triplet hyperfine features due to one or two protons. Triplet features observed using H2O media become doublets using D2O media. Well defined triplet spectra were observed using lithium chloride glasses and methanol–water glasses. Using frozen aqueous solutions which give phase-separated solids, spectra ranging from doublets via mixtures to triplets were obtained, depending on the derivative used. It is suggested that the first formed radical anions are rapidly protonated either on the ring nitrogen [N(3)] or on the amino nitrogen. In the former, there is no extra splitting from the added proton and a major doublet is observed because of hyperfine coupling to the C(6) proton [C(6)–H]. In the latter case, the proton is thought to add along a path normal to the plane of the ring so that overlap with the SOMO is maximised, and there is a large hyperfine coupling. Coupling to the other two amino protons is too small to resolve under these conditions. The fact that the –N(CH3)2 derivative gives a doublet rather than a triplet supports the postulate that the –NH2 group is indeed protonated in the triplet species. For the aqueous-methanol glasses, on annealing, this extra doublet splitting is lost irreversibly, probably because rotation of the –NH+3 group sets in, making the three protons equivalent.As has been shown by Bernhard and co-workers, certain oligomers which give C·– centres on irradiation give rise to doublets only in H2O glasses. Hence N(3)-protonation is favoured in polymeric systems. Reasons for these differences, and the importance of these results in relation to DNA are discussed.

DNA in glasses at 77K : high energy ionizing radiation versus UV electron ejection

Most in the field of ionizing radiation damage to DNA in frozen aqueous solutions agree that two major types of radical ions are formed, i.e. G+/A+ and T-/C-. The main evidence stems from EPR and strand break studies. Fluid solutions exposed to laser light are known to give G+ and esolv- with low yields of single strand breaks. We have explored this contrast by photoionizing DNA solutions at 77 K, in the expectation that this would prevent the formation of esolv- and hence that the results might be similar to those for high energy radiation. They are not: the results show only the formation of G+ (or) (A+), the fate of the ejected electrons is unclear except for sodium perchlorate glasses when they react to give O-.

An EPR study of photoionised thymine and its derivatives at 77 K

The effect of high-energy ionising radiation on thymine and a wide range of its derivatives has been widely studied. Liquid-phase studies on aqueous solutions using pulse radiolysis and UV-photoionisation methods have been reported. Our aim was to use low temperature matrices in combination with laser photolysis and EPR spectroscopy in order to learn more about the double quantum event leading to UV-photoionisation. For thymine, electron-loss results in the normal π-radical cation but this rapidly undergoes proton loss, probably from the N1–H group. The EPR spectrum is well defined and is characteristic of this radical. This species was detected in perchlorate matrices and there was no evidence of electron capture by thymine. However, in pure aqueous systems, TH˙ radicals, possibly formed by electron capture followed by protonation, were detected.In contrast, for N1 substituted species such as TMP, the π-radical cations were not detected by EPR spectroscopy. Instead, there were comparable yields of TCH2˙ radicals, in which one of the methyl protons has been lost. These give rise to a characteristic quartet splitting from hyperfine coupling to the two –CH2˙ protons, together with the C6–H proton which gives about the same splitting. Only in the case of alkaline perchlorate glasses, when the N3–H proton is removed, was the electron lost from the π system to give the neutral π-radical.When frozen aqueous systems containing TCH2˙ radicals were annealed the quartet signals were lost, and quintets grew in. Similar quintets have been previously assigned to TOH˙ radicals formed by the addition of water to TCH2˙. However, use of D2O did not modify the signals, so we propose that this species is due to a dimer radical and/or a cyclic radical. The former is formed by the addition of the TCH2˙ unit to C6 of another molecule and the latter arises from intramolecular hydrogen-atom transfer from the C5′ hydrogen followed by cyclisation.Use of 5-ethyl-2′-deoxyuridine, systems that gave the TCH2· radicals for thymine derivatives, gave UĊHCH3 radicals. Reasons for the formation of these species by biphotonic photoionisation are discussed. It is difficult to understand why the primary π-radical cations of the N1-substituted thymine derivatives should prefer to lose a proton from the carbon rather than the >N3–H groups, since the latter, but not the former, has an adjacent proton-acceptor. An alternative is considered, in which an excited state of the primary electron-loss centre is initially formed. This state has the local structure > CO˙+, the SOMO being the in-plane n-orbital on oxygen. By analogy with, for example, ester radical cations, it is suggested that hydrogen-atom transfer from the adjacent methyl group occurs more rapidly than the switch from this state to the π-ground state, thereby giving the required TCH2˙ centre, with a proton on the adjacent carbonyl group.

Electron addition to DNA-thymine vs cytosine radical anion?

Comparison of the EPR spectra of a self-complementary oligonucleotide (24-mer) containing either thymidine or [6-2H1]thymidine residues following irradiation in frozen aqueous LiCl matrices allows the unambiguous analysis of thte radical anion population in model DNA systems; these studies show that C˙–(1): T˙–(2) is 85 : 15.