The reduction potential of the slipped GC base pair in one-electron oxidized duplex DNA.

Redox equilibrium between the low potential aniline radical cation and the guanine in the GC base pair of duplex DNA has been established using pulse radiolysis. We relate the measurement of a radical one-electron reduction potential, E0', of 1.01 ± 0.03 V to the perturbation of the GC base pair to accommodate the neutral guanyl radical in an energetically more stable 'slipped' structure. The formation of the 'slipped' structure is exothermic by -11.4 kcal mol-1 as calculated by DFT, which is inline with our experimental results.

Characterisation of radicals formed by the triazine 1,4-dioxide hypoxia-activated prodrug, SN30000.

The radical species underlying the activity of the bioreductive anticancer prodrug, SN30000, have been identified by electron paramagnetic resonance and pulse radiolysis techniques. Spin-trapping experiments indicate both an aryl-type radical and an oxidising radical, trapped as a carbon-centred radical, are formed from the protonated radical anion of SN30000. The carbon-centred radical, produced upon the one-electron oxidation of the 2-electron reduced metabolite of SN30000, oxidises 2-deoxyribose, a model for the site of damage on DNA which leads to double strand breaks. Calculations using density functional theory support the assignments made.

Characterization of radicals formed following enzymatic reduction of 3-substituted analogues of the hypoxia-selective cytotoxin 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine).

The mechanism by which the 1,2,4-benzotriazine 1,4-dioxide (BTO) class of bioreductive hypoxia-selective prodrugs (HSPs) form reactive radicals that kill cancer cells has been investigated by steady-state radiolysis, pulse radiolysis (PR), electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. Tirapazamine (TPZ, 3-amino BTO, 1) and a series of 3-substituted analogues, -H (2), -methyl (3), -ethyl (4), -methoxy (5), -ethoxymethoxy (6), and -phenyl (7), were reduced in aqueous solution under anaerobic steady-state radiolysis conditions, and their radicals were found to remove the substrates by short chain reactions of different lengths in the presence of formate ions. Multiple carbon-centered radical intermediates, produced upon anaerobic incubation of the compounds with cytochrome P(450) reductase enriched microsomes, were trapped by N-tert-butyl-alpha-phenylnitrone and observed using EPR. The highly oxidizing oxymethyl radical, from compound 5, was identified, and experimental spectra obtained for compounds 1, 2, 3, and 7 were well simulated after the inclusion of aryl radicals. The identification of a range of oxidizing radicals in the metabolism of the BTO compounds gives a new insight into the mechanism by which these HSPs can cause a wide variety of damage to biological targets such as DNA.

Release of nitrite from the antitubercular nitroimidazole drug PA-824 and analogues upon one-electron reduction in protic, non-aqueous solvent.

The one-electron reduction chemistry of the antituberculosis drug PA-824, together with a series of closely related compounds, has been investigated in irradiated anaerobic propan-2-ol solution. The protic solvent, of low dielectric constant, was chosen to mimic the environment of a water-restricting active site of a model protein, which is capable of reducing the compounds. Radiolytic reduction of the compounds containing electron donating substituents in the 2-position of the imidazole ring released nitrite, with compounds that are highly active against Mycobacterium tuberculosis exhibiting high yields of nitrite. The release of cytotoxic reactive nitrogen species through a one-electron pathway, by as yet unidentified proteins, may play a role in the activity of this class of compounds against TB. The described radiolytic quantification of nitrite release may have utility as a preliminary screening test for nitroaromatic candidate drugs against the disease.

One-electron reduction potential of the neutral guanyl radical in the GC base pair of duplex DNA.

The one-electron oxidation of guanine in the GC base pair of DNA has been investigated using pulse radiolysis combined with DFT calculations. Reaction of benzotriazinyl radicals with DNA results in the formation of the neutral guanyl radical and redox equilibria. The one-electron reduction potential, E(7), of the neutral guanyl radical in the GC base pair is determined for the first time as 1.22 +/- 0.02 V, from both absorption and kinetic data.

Pulse radiolysis investigation on the mechanism of the catalytic action of Mn(II)-pentaazamacrocycle compounds as superoxide dismutase mimetics.

The mechanism for the catalytic dismutation of superoxide by the Mn(II) pentaazamacrocyclic compound M40403 ([manganese(II) dichloro-(4 R,9 R,14 R,19 R)-3,10,13,20,26 pentaazatetracyclo [20.3.1.0 (4,9).0 (14,9)] hexacosa-1(26),-22(23),24-triene], SODm1) and two 2,21-dimethyl analogues has been investigated using pulse radiolysis. The initial rate of reaction between superoxide and the manganese compounds was found to be dependent on structure and pH, with the resulting transient adducts possessing spectral characteristics of the metal center being oxidized to Mn(III). Values for the p K a of the transient adducts (p K a = 5.65 +/- 0.05; 5.3 +/- 0.1 and <5 for SODm1, SODm2 and SODm3, respectively) were obtained from spectrophotometric and conductivity measurements. Reaction of these transient adducts with further superoxide was highly structure dependent with the 2 S,21 S-dimethyl derivative (SODm2) being highly catalytically active at pH 7.4 ( k cat = 2.35 x 10 (8) M (-1) s (-1)) compared to SODm1 ( k cat = 3.55 x 10 (6) M (-1) s (-1)). In contrast the 2 R,21 R-dimethyl derivative (SODm3) showed no dismutation catalysis at all. The reaction rates of the initial complexes with HO 2 (*) were significantly lower than with O 2 (*-), and it is proposed that O 2 (*-) is the main reactant in the catalytic cycle at pH 7.4. Variable temperature studies revealed major differences in the thermodynamics of the catalytic cycles involving SODm2 or SODm1. In the case of SODm2, the observed high entropic contribution to the activation energy is indicative of ligand conformational changes during the catalytic step. These results have provided the basis for a new mechanism for the catalytic dismutation of superoxide by Mn(II)-pentaazamacrocycle SOD mimetics.

A mitochondria-targeted macrocyclic Mn(II) superoxide dismutase mimetic.

Superoxide (O(2)(·-)) is the proximal mitochondrial reactive oxygen species underlying pathology and redox signaling. This central role prioritizes development of a mitochondria-targeted reagent selective for controlling O(2)(·-). We have conjugated a mitochondria-targeting triphenylphosphonium (TPP) cation to a O(2)(·-)-selective pentaaza macrocyclic Mn(II) superoxide dismutase (SOD) mimetic to make MitoSOD, a mitochondria-targeted SOD mimetic. MitoSOD showed rapid and extensive membrane potential-dependent uptake into mitochondria without loss of Mn and retained SOD activity. Pulse radiolysis measurements confirmed that MitoSOD was a very effective catalytic SOD mimetic. MitoSOD also catalyzes the ascorbate-dependent reduction of O(2)(·-). The combination of mitochondrial uptake and O(2)(·-) scavenging by MitoSOD decreased inactivation of the matrix enzyme aconitase caused by O(2)(·-). MitoSOD is an effective mitochondria-targeted macrocyclic SOD mimetic that selectively protects mitochondria from O(2)(·-) damage.

Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302.

TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect.

Spectral and kinetic evidence for reaction of superoxide with compound I of myeloperoxidase.

Superoxide and myeloperoxidase (MPO) are essential for the oxidative killing of bacteria by neutrophils. Previously, we developed a kinetic model to demonstrate that within the confines of neutrophil phagosomes, superoxide should react exclusively with MPO and be converted to hypochlorous acid. The model consists of all known reactions and rate constants for reactions of superoxide, hydrogen peroxide, and chloride ions with MPO, except for the reaction of superoxide with compound I, which could only be estimated. Compound I is a transitory redox intermediate of MPO that is responsible for oxidizing chloride ions to hypochlorous acid. To tackle the challenge of observing the reaction between two transient species, we combined stopped-flow spectrophotometry with pulse radiolysis. Using this technique, we directly observed the reduction of compound I by superoxide. The rate constant for the reaction was determined to be 5.6±0.3×10(6)M(-1)s(-1). This value establishes superoxide as one of the best substrates for compound I. Based on this value, the rate constant for reduction of compound II by superoxide was determined to be 1.2±0.1×10(6)M(-1)s(-1). Within phagosomes, the reduction of compound I by superoxide will compete with the oxidation of chloride ions so that the relative concentrations of these two substrates will affect the yield of hypochlorous acid. Characterization of this reaction confirms that superoxide is a physiological substrate for MPO and that their interactions are central to an important host defense mechanism.

Reactivity of ubiquinone and ubiquinol with superoxide and the hydroperoxyl radical: implications for in vivo antioxidant activity.

Endogenous ubiquinones (UQ) such as coenzyme Q(10) are essential electron carriers in the mitochondrial respiratory chain, and the reduced ubiquinol form (UQH(2)) is a chain-breaking antioxidant, decreasing oxidative damage caused by lipid peroxidation within mitochondria. Consequently, exogenous UQ are used as therapies to decrease mitochondrial oxidative damage. The proximal radical produced during mitochondrial oxidative stress is superoxide (O(2)(.-)) and the reaction between UQ and O(2)(.-) to form the ubisemiquinone radical anion (UQ(.-)) may also be important for the scavenging of O(2)(.-) by exogenous UQ. The situation in vivo is that many UQ are predominantly located in the hydrophobic membrane core, from which O(2)(.-) will be excluded but its conjugate acid, HOO(.), can enter. The reactivity of UQ or UQH(2) with HOO(.) has not been reported previously. Here a pulse radiolysis study on the reactions between UQ/UQH(2) and O(2)(.-)/HOO(.) in water and in solvent systems mimicking the surface and core of biological membranes has been undertaken. O(2)(.-) reacts very rapidly with UQ, suggesting that this may contribute to the scavenging of O(2)(.-) in vivo. In contrast, UQH(2) reacts relatively slowly with HOO(.), but rapidly with other oxygen- and carbon-centered radicals, indicating that the antioxidant role of UQH(2) is mainly in preventing lipid peroxidation.

Intermediates in the reduction of the antituberculosis drug PA-824, (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine, in aqueous solution.

The reduction chemistry of the new anti-tuberculosis drug PA-824, together with a more water-soluble analogue, have been investigated using pulse and steady-state radiolysis in aqueous solution. Stepwise reduction of these nitroimidazo-dihydrooxazine compounds through electron transfer from the CO(2) (-) species revealed that, unlike related nitroimidazoles, 2-electron addition resulted in the reduction of the imidazole ring in preference to the nitro group. In mildly acidic solution a nitrodihydroimidazo intermediate was formed, which was reduced further to the amine product. In both alkaline and neutral solution, an intermediate produced on 2-electron reduction was resistant to further reduction and reverted to parent compound on extraction or mass spectrometric analysis of the solution. The unusual reduction chemistry of these nitroimidazole compounds, exhibiting ring over nitro group reduction, is associated with alkoxy substitution in the 2-position of a 4-nitroimidazole. The unique properties of the intermediates formed on the reduction of PA-824 need to be considered as playing a possible role in its bactericidal action.

Cytosine-gated hole creation and transfer in DNA in aqueous solution.

The selenite radical, SeO3-, has been found to selectively produce the cytosyl radical upon one-electron oxidation of duplex DNA. This is at first a surprising result as SeO3- can only oxidize guanine of the DNA bases, implying that the transiently formed guanyl radical cation must transpose into the neutral cytosyl radical with loss of a proton. Back oxidation to produce the neutral guanyl radical, in competition with another fixation reaction, is observed.

Ionization of aniline and its N-methyl and N-phenyl substituted derivatives by (free) electron transfer to n-butyl chloride parent radical cations.

The electron transfer from aniline and its N-methyl as well as N-phenyl substituted derivatives (N-methylaniline, N,N-dimethylaniline, diphenylamine, triphenylamine) to parent solvent radical cations was studied by electron pulse radiolysis in n-butyl chloride solution. The ionization results in the case of aniline (ArNH2) and the secondary aromatic amines (Ar2NH, Ar(Me)NH) in the synchronous and direct formation of amine radical cations, as well as aminyl radicals, in comparable amounts. Subsequently, ArNH2*+ deprotonates in a delayed reaction with the present nucleophile Cl-, and forms further ArNH*. In contrast, tertiary aromatic amines such as triphenylamine and dimethylaniline yield primarily the corresponding amine radical cations Ar3N*+ or Ar(Me2)N*+, only. The persistent Ar3N*+ forms a charge transfer complex (dimer) with the parent amine molecule, whereas Ar(Me2)N*+ deprotonates to carbon-centered radicals Ar(Me)NCH2*.

Ionization of cyclic aromatic amines by free electron transfer: products are governed by molecule flexibility.

The bimolecular electron transfer from secondary aromatic amines to parent radical cations of nonpolar solvents such as alkanes and alkyl chlorides results in the synchronous formation of amine radical cations as well as aminyl radicals, in comparable amounts. If as for cyclic aromatic amines (c-Ar(2)NH) the intramolecular bending motion around the amine group is restricted in varying degrees (acridane, phenothiazine) or completely prevented (carbazole), then this picture is modified. In the free electron transfer, the completely rigid carbazole yields exclusively amine radical cations. Acridane exhibits preferred radical cations, but phenothiazine with the more flexible six-membered ring involving sulfur as a further heteroatom follows the common two-product rule; see above. The phenomenon is reasoned by a peculiarity in the bimolecular free electron transfer where after diffusional approach the actual electron jump proceeds in the ultrashort time range. Therefore, it reflects femtosecond molecular motions which, in the case of free mobility, continuously pass through different molecule conformers, combined with fluctuation of the electrons of the responsible molecular n-orbitals. The rigid systems, however, do not show this effect because of a nonexistent bending motion.