Studies on reactions of oxidizing sulfur–sulfur three-electron-bond complexes and reducing α-amino radicals derived from OH reaction with methionine in aqueous solution

The technique of pulse radiolysis with spectrophotometric detection has been used to investigate the possibility of electron transfer reactions between oxidizing sulfur–sulfur three-electron-bond complexes (Met₂/S∴S⁺), or reducing α-amino radicals (CH₃SCH₂CH₂CH^⋅NH₂) derived from reaction of methionine with OH radicals and hydroxycinnamic acid (HCA) derivatives, riboflavin (RF) or flavin adenine dinucleotide (FAD), respectively. The HCA derivatives, such as caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, widely distributed phenolic acids in fruit and vegetables, have been identified as good antioxidants previously can rapidly and efficiently repair oxidizing three-electron-bond complexes via electron transfer. RF and FAD can oxidize reducing α-amino radicals derived from methionine. The electron transfer rate constants ∼10⁹ dm³ mol⁻¹ s⁻¹ were determined by following the build-up kinetics of species produced.     

Repair of oxidative DNA damage by amino acids

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)₂^–, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of ~10⁵, 10⁵, 10⁶ and 10⁷ dm³ mol^–1 s^–1, respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.     

Peptide repair of oxidative DNA damage

Guanyl radical species are produced in DNA by electron removal caused by ionizing radiation, photoionization, oxidation, or photosensitization. DNA guanyl radicals can be reduced by electron donation from mild reducing agents. Important biologically relevant examples are the redox active amino acids cysteine, cystine, methionine, tryptophan, and tyrosine. We have quantified the reactivity of derivatives of these amino acids with guanyl radicals located in plasmid DNA. The radicals were produced by electron removal using the single electron oxidizing agent (SCN)(2)(*)(-). Disulfides (cystine) are unreactive. Thioethers (methionine), thiols (cysteine), and phenols (tyrosine) react with rate constants in the range 10(4)-10(6), 10(5)-10(6), and 10(5)-10(6) dm(3) mol(-1) s(-1), respectively. Indoles (tryptophan) are the most reactive with rate constants of 10(7)-10(8) dm(3) mol(-1) s(-1). Selenium analogues of amino acids are over an order of magnitude more reactive than their sulfur equivalents. Increasing positive charge is associated with a ca. 10-fold increase in reactivity. The results suggest that amino acid residues located close to DNA (for example, in DNA binding proteins such as histones) might participate in the repair of oxidative DNA damage.     

Reaction of reducing hydroxyl radical adducts of pyrimidine nucleotides with riboflavin and flavin adenine dinucleotide (FAD) via electron transfer: a pulse radiolysis study

Using the techniques of pulse radiolysis with time-resolved spectrophotometric detection, it has been demonstrated that the interaction of reducing OH radical adducts of dCMP, TMP and UMP with riboflavin (RF) and flavin adenine dinucleotide (FAD) does proceed via an electron transfer reaction. From buildup kinetics of radical species, the rate constants of electron transfer from reducing OH adducts of pyrimidines to RF and FAD have been determined, respectively. It could be deduced that RF and FAD would reduce the probability of repair of the damaged DNA in the presence of enzymes and antioxidants, accordingly RF and FAD might have a radiosensitization effect on DNA damage.     

Radical production from free and peptide-bound methionine sulfoxide oxidation by peroxynitrite and hydrogen peroxide/iron(II)

Methionine sulfoxide is a post-translational protein modification that has been receiving increasing attention in the literature. Here we used electron paramagnetic resonance spin trapping techniques to show that free and peptide-bound methionine sulfoxide is oxidized by hydrogen peroxide/iron(II)-EDTA and peroxynitrite through the intermediacy of the hydroxyl radical to produce both *CH3 and *CH2CH2CH radicals. The results indicate that methionine sulfoxide residues are important targets of reactive oxygen- and nitrogen-derived species in proteins. Since the produced protein-derived radicals can propagate oxidative damage, the results add a new antioxidant route for the action of the enzyme peptide methionine sulfoxide reductase.     

The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study.

Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

The roles of thiol-derived radicals in the use of 2',7'-dichlorodihydrofluorescein as a probe for oxidative stress

2',7'-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are "repaired" by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2',7'-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of approximately 4 or approximately 2x10(7) M(-1) s(-1), respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH* absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS* and CysS* toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS*. DCF formation from interaction of DCFH2 with GS* was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables--including pH and oxygen concentration--which control thiyl radical chemistry.     

Electron transfer in human methionine synthase reductase studied by stopped-flow spectrophotometry

Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s(-1)) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s(-1)) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s(-1)) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-2H]NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP+. The observed rate constant of reverse hydride transfer increases hyperbolically with NADP+ concentration with the FAD domain. The stopped-flow kinetic data, in conjunction with the reported redox potentials of the flavin cofactors for MSR [Wolthers, K. R., Basran, J., Munro, A. W., and Scrutton, N. S. (2003) Biochemistry, 42, 3911-3920], are used to define the mechanism of electron transfer for the reductive half-reaction of MSR. Comparisons are made with similar stopped-flow kinetic studies of the structurally related enzymes cytochrome P450 reductase and nitric oxide synthase.     

Tryptophan 697 modulates hydride and interflavin electron transfer in human methionine synthase reductase

Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supports FAD to FMN electron transfer. Accelerated rates of FAD reduction, steady-state cytochrome c(3+) turnover, and uncoupled NADPH oxidation in the S698Δ and W697H mutants may be attributed to a decrease in the energy barrier for displacement of W697 by NADPH. Binding of NADP(+), but not 2',5'-ADP, is tighter for all mutants than for native MSR. The combined studies demonstrate that while W697 attenuates hydride transfer, it ensures coenzyme selectivity and accelerates FAD to FMN electron transfer. Moreover, analysis of analogous cytochrome P450 reductase (CPR) variants points to key differences in the driving force for flavin reduction and suggests that the conserved FAD stacking tryptophan residue in CPR also promotes interflavin electron transfer.     

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.     

A catalytic intermediate and several flavin redox states stabilized by folate-dependent tRNA methyltransferase from Bacillus subtilis

The flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH(2)THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH(?) radical, and a catalytic intermediate endowed with methylating activity. The FADH(?) radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH(2)THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH(2)THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH(2)THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions.     

Double roles of hydroxycinnamic acid derivatives in protection against lysozyme oxidation

Oxidative damage to protein has been implicated in a number of diseases. Much interest has been focused on preventing oxidative damage to protein. Here we showed that hydroxycinnamic acid derivatives (HCA) were able to inhibit the cross-linking of protein induced by riboflavin-mediated photooxidation. HCA were also found to strongly protect lysozyme from gamma rays irradiation. The antioxidative properties of HCA were further studied by laser flash photolysis. Mechanism of antioxidant activities of HCA on lysozyme oxidation was discussed. HCA were found to protect protein against oxidation by scavenging oxidizing species and repairing the damaged protein.     

Theoretical analysis of the deactivation reactions of triplet state riboflavin by hydroxycinnamic acid derivatives

A thermodynamic analysis of the deactivation reactions of triplet state riboflavin (RF) by hydroxycinnamic acid derivatives has been performed on the basis of quantum chemical calculations. It was revealed that the H-atom transfer pathway is more thermodynamically feasible in comparison with the direct energy/electron transfer to be involved in the triplet state RF quenching by hydroxycinnamic acid derivatives. The results provide some deeper insights into the protective behaviours of hydroxycinnamic acid derivatives against the RF induced photooxidative damage.     

Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O > NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.     

Structure-dependent reactivity of oxyfunctionalized acetophenones in the photooxidation of DNA: base oxidation and strand breaks through photolytic radical formation (spin trapping, EPR spectroscopy, transient kinetics) versus photosensitization (electron transfer, hydrogen-atom abstraction)

The photooxidative damage of DNA, specifically guanine oxidation and strand-break formation, by sidechain-oxyfunctionalized acetophenones (hydroxy, methoxy, tert-butoxy and acetoxy derivatives), has been examined. The involvement of triplet-excited ketones and their reactivity towards DNA has been determined by time-resolved laser-flash spectroscopy. The generation of carbon-centered radical species upon Norrish-type I cleavage has been assessed by spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide, coupled with electron paramagnetic resonance spectroscopy. The observed DNA-base oxidation and strand-break formation is discussed in terms of the peroxyl radicals derived from the triplet-excited ketones by α cleavage and molecular oxygen trapping, as well as direct interaction of the excited states by electron transfer and hydrogen-atom abstraction. It is concluded that acetophenone derivatives, which produce radicals upon photolysis, in particular the hydroxy (AP-OH) and tert-butoxy (AP-O^tBu) derivatives, are more effective in oxidizing DNA.     

Effect of phosphate ions on the fluorescence of tryptophan derivatives. Implications in fluorescence investigation of protein-nucleic acid complexes

In order to test the ability of phosphate groups to quench the fluorescence of tryptophan in protein-nucleic acid complexes we have studied the effect of various phosphate ions on the fluorescence of tryptophan derivatives. Unsubstituted and monoalkyl monoanions (H2PO4- and CH3OPO3H-) quench the fluorescence of all investigated indole derivatives while the dimethyl anion (CH3O)2 PO2- does not. This suggests that quenching of tryptophan fluorescence by phosphate monoanions requires the presence of an acidic OH group and could be due to a proton transfer from the phosphate ion to the indole chromophore. Trianions (PO4 3-4) which are strong proton acceptors quench the fluorescence of all tryptophan derivatives except N(1)methyl tryptophan. This result strongly supports our proposal that quenching of tryptophan fluorescence by phosphate trianions occurs through deprotonation of the NH indole group. Bianions (HPO '4(7), and CH3O PO3 2-3) quench the fluorescence of several indole derivatives including N-acetyl tryptophanamide but have no effect on tryptophan or N(1)-methyl tryptophan. From our results we conclude that phosphate groups of nucleic acids are not able to quench the fluorescence of tryptophyl residues in protein-nucleic acid complexes except if an accessible residue is located near a phosphorylated polynucleotide chain end.     

E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydrated electron.

The reactions of hydrated electrons (eaq-) with amino acids were investigated by the spin-trapping method and by electron spin resonance. Tertiary nitrosobutane was used as a spin-trap to stabilize the short-lived radicals. Hydrated electrons were produced by gamma-radiolysis of de-aerated aqueous solutions of amino acids in the presence of sodium formate or tertiary butanol to scavenge OH. Radicals produced by reductive deamination of 19 amino acids were identified. Radicals formed by scission of the CH3-S- and -S-CH2- bonds of methionine as well as by deamination were observed. In the case of phenylalanine the radical formed by electron addition followed by proton transfer was identified. The reaction of proline and of hydroxyproline with eaq- resulted in the opening of the cyclic structure.     

Interflavin one-electron transfer in the inducible nitric oxide synthase reductase domain and NADPH-cytochrome P450 reductase

In this study, we have analyzed interflavin electron transfer reactions from FAD to FMN in both the full-length inducible nitric oxide synthase (iNOS) and its reductase domain. Comparison is made with the interflavin electron transfer in NADPH-cytochrome P450 reductase (CPR). For the analysis of interflavin electron transfer and the flavin intermediates observed during catalysis we have used menadione (MD), which can accept an electron from both the FAD and FMN sites of the enzyme. A characteristic absorption peak at 630 and 520 nm can identify each FAD and FMN semiquinone species, which is derived from CPR and iNOS, respectively. The charge transfer complexes of FAD with NADP+ or NADPH were monitored at 750 nm. In the presence of MD, the air-stable neutral (blue) semiquinone form (FAD-FMNH*) was observed as a major intermediate during the catalytic cycle in both the iNOS reductase domain and full-length enzyme, and its formation occurred without any lag phase indicating rapid interflavin electron transfer following the reduction of FAD by NADPH. These data also strongly suggest that the low level reactivity of a neutral (blue) FMN semiquinone radical with electron acceptors enables one-electron transfer in the catalytic cycle of both the FAD-FMN pairs in CPR and iNOS. On the basis of these data, we propose a common model for the catalytic cycle of both CaM-bound iNOS reductase domain and CPR.     

Site of OH radical attack on dihydrouracil and some of its methyl derivatives

The site of attack of OH radicals on dihydrouracil and five of its methylated derivatives was determined by pulse radiolysis using N,N,N',N'-tetramethylphenylenediamine (TMPD) to detect oxidizing radicals and tetranitromethane (TNM) as well as K3Fe(CN)6 to detect reducing radicals. In the case of dihydrouracil OH radicals abstract preferentially an H atom at C(6) to give the 6-yl radical (greater than or equal to 90 per cent) which at pH approximately 6.5 reduces TNM and K3Fe(CN)6 at almost diffusion-controlled rates. Only a small fraction of OH radicals abstract the H atom at C(5) (less than or equal to 10 per cent). The resulting 5-yl radical oxidizes TMPD to TMPD+ at pH 7-8. With the methylated derivatives of dihydrouracil, OH radicals react less selectively, especially in the case of N(1)-methyl derivatives. This methyl group is activated to a similar degree as the methylene group at C(6). In 1-Medihydrouracil the yield of N(1)-CH2 radicals is about 29 per cent, which has been deduced from the yield of formaldehyde formed after oxidation of this radical by TNM at pH approximately 6.5 and the subsequent hydrolysis. Radicals at the other methyl substituents are generated to a lesser extent (less than or equal to 10 per cent) and are relatively unreactive towards oxidizing agents such as TNM and K3Fe(CN)6 as well as towards the reducing agent TMPD. Although methyl substitution opens new routes for OH attack the preferred site of H abstraction remains C(6) (greater than 60 per cent).