Kantilal B. Patel

Free hydroxyl radicals are formed on reaction between the neutrophil-derived species Superoxide anion and hypochlorous acid

Superoxide anion reacts with hypochlorous acid to yield free hydroxyl radicals, as shown by the hydroxylation of benzoate. This reaction is analogous to the Haber‐Weiss reaction but in the absence of metal ions is at least six orders of magnitude faster.

Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate.

One-electron oxidation of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) by the azide radical generates the radical cation (H(4)B(*)(+)) which rapidly deprotonates at physiological pH to give the neutral trihydrobiopterin radical (H(3)B(*)); pK(a) (H(4)B(*)(+) <==> H(3)B(*) + H(+)) = (5.2 +/- 0.1). In the absence of ascorbate both the H(4)B(*)(+) and H(3)B(*) radicals undergo disproportionation to form quinonoid dihydrobiopterin (qH(2)B) and the parent H(4)B with rate constants k(H(4)B(*)(+) + H(4)B(*)(+)) = 6.5 x 10(3) M(-1) s(-1) and k(H(3)B(*) + H(3)B(*)) = 9.3 x 10(4) M(-1) s(-1), respectively. The H(3)B(*) radical is scavenged by ascorbate (AscH(-)) with an estimated rate constant of k(H(3)B(*) + AscH(-)) similar 1.7 x 10(5) M(-1) s(-1). At physiological pH the pterin rapidly scavenges a range of biological oxidants often associated with cellular oxidative stress and nitric oxide synthase (NOS) dysfunction including hydroxyl ((*)OH), nitrogen dioxide (NO(2)(*)), glutathione thiyl (GS(*)), and carbonate (CO(3)(*-)) radicals. Without exception these radicals react appreciably faster with H(4)B than with AscH(-) with k(*OH + H(4)B) = 8.8 x 10(9) M(-1) s(-1), k(NO(2)(*) + H(4)B) = 9.4 x 10(8) M(-1) s(-1), k(CO(3)(*-) + H(4)B) = 4.6 x 10(9) M(-1) s(-1), and k(GS(*) + H(4)B) = 1.1 x 10(9) M(-1) s(-1), respectively. The glutathione disulfide radical anion (GSSG(*-)) rapidly reduces the pterin to the tetrahydrobiopterin radical anion (H(4)B(*-)) with a rate constant of k(GSSG(*-) + H(4)B) similar 4.5 x 10(8) M(-1) s(-1). The results are discussed in the context of the general antioxidant properties of the pterin and the redox role played by H(4)B in NOS catalysis.

Pitfalls in the Use of Common Luminescent Probes for Oxidative and Nitrosative Stress

Lucigenin (LC2+, bis-N-methylacridinium) and 2′,7′-dichlorofluorescin (DCFH2) are widely used as chemiluminescent or fluorescent probes for cellular oxidative stress, to reflect levels of superoxide (O2·−) and hydrogen peroxide, respectively. We report mechanistic studies that add to the growing evidence for the unsuitability of either probe except in very well-defined circumstances. The ability for lucigenin to generate superoxide via reduction of LC2+ to LC·+ and redox cycling with oxygen depends on the reduction potential of the LC2+/LC·+ couple. Redox equilibrium between LC·+ and the redox indicator benzyl viologen is established in microseconds after generation of the radicals by pulse radiolysis and indicated E(LC2+/LC·+) ∼ −0.28 V vs. NHE. Reaction of LC·+ with O2 to generate O2·− was also observed directly similarly, occurring in milliseconds, with a rate constant k ∼ 3 × 106M−1 s−1. Quinones act as redox mediators in LC·+/O2 redox cycling. Oxidation of DCFH2 to fluorescent DCF is not achieved by O2·− or H2O2, but NO2·) reacts rapidly: k ∼ 1 × 107M−1 s−1. Oxidation by H2O2 requires a catalyst: cytochrome c (released into the cytosol in apoptosis) is very effective (even 10 nM). Fluorescence reflects catalyst level as much as O2·−) production.

Bioreductively-activated prodrugs for targeting hypoxic tissues: Elimination of aspirin from 2-nitroimidazole derivatives

2-Nitroimidazoles were synthesised substituted with aspirin or salicylic acid, as leaving groups linked through the (imidazol-5-yl)methyl position. Activation of aqueous solutions by CO2*- (a model one-electron reductant) resulted in release of aspirin or salicylate, probably via the 2-hydroxyaminoimidazole. The analogous 2-nitroimidazole with bromide as leaving group eliminated bromide in < 1 ms via the radical-anion.

Synthesis and biological properties of bioreductively targeted nitrothienyl prodrugs of combretastatin A-4

Nitrothienylprop-2-yl ether formation on the 3′-phenolic position of combretastatin A-4 (1) abolishes the cytotoxicity and tubulin polymerization-inhibitory effects of the drug. 5-Nitrothiophene derivatives of 1 were synthesized following model kinetic studies with analogous coumarin derivatives, and of these, compound 13 represents a promising new lead in bioreductively targeted cytotoxic anticancer therapies. In this compound, optimized gem-dimethyl α-carbon substitution enhances both the aerobic metabolic stability and the efficiency of hypoxia-mediated drug release. Only the gem-substituted derivative 13 released 1 under anoxia in either in vitro whole-cell experiments or supersomal suspensions. The rate of release of 1 from the radical anions of these prodrugs is enhanced by greater methyl substitution on the α-carbon. Cellular and supersomal studies showed that this α-substitution pattern controls the useful range of oxygen concentrations over which 1 can be effectively released by the prodrug. [Mol Cancer Ther 2006;5(11):2886–94]

Pulse radiolysis studies of electron migration in DNA from DNA base-radical anions to nitroacridine intercalators in aqueous solution

The reactions of the aquated electron (e–aq) with intercalators of high reduction potential (nitracrine and related basic nitroacridines) has been investigated by pulse radiolysis in the presence of DNA in aqueous solution. Under conditions where the majority of the e–aq species react initially with DNA bases (high DNA: drug ratios) a slower subsequent electron transfer to the intercalator was observed. The rate of this intra-complex transfer, expressed as DNA base pairs traversed per second, was in the range (1.2–3.1)× 105 base pairs s–1 and increased in order of the one-electron reduction potentials of the DNA-bound intercalators. No transfer was seen to the much less electron affinic des-nitro analogue of the nitroacridines. Only a small proportion of the initial DNA base radicals (⩽50%) underwent this intra-complex electron transfer. Even for the most efficient electron trap, nitracrine, the apparent mean electron migration distance was only three base pairs. A slow secondary reduction of nitroacridines [(0.08–5.0)× 104 base pairs s–1] was also observed with a proportion of the essentially immobile ˙OH-induced DNA radicals. This secondary reaction may well serve as a measure of the mobility of the DNA-bound intercalators. This study therefore implies a lack of extensive migration of DNA-associated electrons in aqueous solution, although it does not exclude the possibility that more mobile electrons produced by direct ionization of DNA might migrate over large distances.

Prodrugs for targeting hypoxic tissues : Regiospecific elimination of aspirin from reduced indolequinones

A series of regioisomeric derivatives of a 1-methylindole-4,7-dione were synthesised, substituted with a 2-acetoxybenzoate leaving group linked through the (indol-2-yl)methyl or (indol-3-yl)methyl (or propenyl) positions. Reductive elimination of the leaving group occurred from the (indol-3-yl)methyl derivatives but not the 2-substituted regioisomers, indicating that only the C-3 position may be utilised in bioreductively-activated drug delivery, which was demonstrated with an aspirin prodrug.

Radical spectra and product distribution following electrophilic attack by the OH˙ radical on 4-hydroxybenzoic acid and subsequent oxidation

The distribution of electrophilic OH˙ radical addition to 4-hydroxybenzoic acid (HBA) has been determined by oxidizing the radical intermediates (substituted hydroxycyclohexadienyl radicals) with quinones and viologens, to yield products. It is deduced from product analysis using high-performance liquid chromatography that the fractions of OH˙ attack at the 1 : 2 : 3 : 4 positions of HBA are 0.16 : 0.04 : 0.65 : 0.15 respectively. Pulse radiolysis studies show that the rate of electron transfer from the radical intermediate formed by OH˙ addition to position 3 of HBA is dependent on the one-electron reduction potential of the oxidant. This electron-transfer process is in competition with the elimination of water by general acid–base catalysis to yield the phenoxyl radical. Catalysis by OH– proceeds through the formation of the deprotonated species, pKa= 8.4 ± 0.2, followed by the elimination of water, Ke= 3.0 ± 0.3 × 105 s–1. The addition of an OH˙ radical to position 3 of HBA gives rise to an absorption band centred at 365 nm.

Radical cations of some low-potential viologen compounds. Reduction potentials and electron-transfer reactions

The one-electron reduction potentials (E1) of certain pyrazinediium, diazepinediium and diazocinediium viologen compounds substituted with methyl groups, V2+, have been determined from the position of the one-electron transfer equilibria with reference compounds using pulse radiolysis. E1 ranges from –491 ± 6 mV (vs NHE) for 6,7-dihydro-2,11-dimethyldipyrido-[1,2-a : 2′,1′-c]pyrazinediium dibromide (V212+) to –832 ± 11 mV for 6,7,8,9-tetrahydro-2,3,12,13-tetramethyldipyrido[1,2-a : 2′,1′-c][1,4]diazocinediium dibromide (V422+).The rates of reduction by e–aq were found to be independent of E1 whereas the rates of electron transfer from CO˙–2 and (CH3)2ĊOH species do show a dependence on E1 for V2+ compounds of lowest E1. Marcus-type treatment of the rate-constant data yields a rate constant of electron exchange between propan-2-oxyl radicals and acetone in the range (2–6)× 106 dm3 mol–1 s–1 and between the radical cation of 6,7-dihydro-2,3,10,11-tetramethyldipyrido[1,2-a : 2′,1′-c]-pyrazinediium dibromide (V22˙+) and its unreduced form (V222+) of 5 × 107 dm3 mol–1 s–1.

Critical Residues in D-amino Acid Oxidase a Pulse-radiolysis and Inactivation Study

The enzyme D-amino acid oxidase and its apoenzyme have been irradiated at pH 5.5--10 under conditions designed to assess the inactivating effect of OH radicals and the selective free radicals Br2- and (SCN)2-. Near neutral pH, removal of the coenzyme FAD from the enzyme results in greater inactivation by selective free-radical attack. From pulse-radiolysis spectra, this increase is associated with attack on tyrosine and tryptophan residues in the protein. A large increase in inactivation of both the haloenzyme and apoenzyme by selective free-radical attack is seen with increasing alkalinity. This is consistent with attack on tyrosine being of major importance.