Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a very promising anticancer drug, already approved for clinical use in three therapeutic indications. It is metabolized intracellularly to 5'-diphosphate (dFdCDP), which is known to be a potent inhibitor of ribonucleotide reductase (RNR). Although several nucleotide analogs show in vitro capacity of RNR inactivation, none has shown the in vivo efficacy of dFdCDP. Accordingly, the experimental data suggests that its mechanism of inhibition is different from the other known RNR suicide inhibitors. Enzyme inhibition in the absence of reductive species leads to complete loss of the essential radical in subunit R2, and formation of a new nucleotide-based radical. Interestingly, however, the presence of the reductants does not prevent inhibition--the radical is not lost but the targeted subunit of RNR becomes R1, which is inactivated possibly by alkylation. We have conducted a theoretical study, which led us to the first proposal of a possible mechanism for RNR inhibition by dFdCDP in the absence of reductants. This mechanism turned out to be very similar to the natural substrate reduction pathway and only deviates from the natural course after the formation of the well-known disulphide bridge. This deviation is caused precisely by the F atom in the beta-face, only present in this inhibitor. The essential radical in R2 is lost, and so is the enzyme catalytic activity. The nucleotide-based radical that constitutes the end product of our mechanism has been suggested in the literature as a possible candidate for the one detected experimentally. In fact, all experimental data available has been reproduced by the theoretical calculations performed here.     

Understanding ribonucleotide reductase inactivation by gemcitabine

This paper focuses on the inhibition of ribonucleotide reductase (RNR) by gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), a deoxycytidine analogue that is a very active drug against solid tumors and is currently commercialized as gemzar. RNR inactivation is reductant-dependent and occurs in a very different way from that of other known substrate analogues. In the presence of reductants monomer R1 of RNR is inhibited, whereas in the absence of reductants the radical is lost and monomer R2 is inhibited. As inside the cell reductants are available, it is likely that R1 inactivation is the most favorable mechanism responsible for drug cytotoxicity. This inhibition pathway has been unknown to date, but we have conducted a theoretical study that has led us to the first proposal of a mechanism for RNR inhibition by dFdC in the presence of reductants.     

Enzyme ribonucleotide reductase: unraveling an enigmatic paradigm of enzyme inhibition by furanone derivatives

Several 2'-substituted-2'-deoxyribonucleotides are potent inactivators of the enzyme ribonucleotide reductase (RNR), by destroying the essential tyrosyl radical located in subunit R2 or/and covalently alkylating the subunit R1. In the absence of external reductants, the inactivation is achieved by alkylation of subunit R1 by a methylene-3(2H)-furanone. The furanone is generated in solution through degradation of a keto-deoxyribonucleotide intermediate, produced during the inhibitory mechanism of a wide group of 2'-substituted inhibitors, and is easily detected experimentally by UV spectroscopy. Interestingly, the same keto-deoxyribonucleotide is also a proposed intermediate of the normal substrate pathway, but by some unknown reason, it does not dissociate from the active site and does not inactivate the enzyme. Therefore, if the currently accepted mechanism for substrate reduction is correct, there must be some specific reason that makes such a reactive intermediate behave differently, not dissociating from the active site during substrate reduction. In this article, we propose to validate the current substrate mechanism by showing that the keto-deoxyribonucleotide dissociates from the active site only in the case of the inhibitors, and therefore, it corresponds to a viable intermediate in the substrate mechanism. Furthermore, we answer unexplained experimental observations that concern the predomination of the normal reduction mechanism over the abnormal ketone formation in the FdNDP and the release of F(-), either in the normal or in the abnormal turnover. For that purpose, we have investigated the interaction between the enzyme and this keto-deoxyribonucleotide generated from the normal substrate and from two widely studied representative inhibitors. A model containing 140 atoms was used to represent the desired structures. The results allowed us to conclude that the solvation free energy of the 2'-substituents, its influence inside the active site, and the charge transfer mechanism from a protein side chain to solution are the thermodynamic driving forces for the intermediate dissociation and subsequent RNR inhibition. Such charge transfer cannot be accomplished by the natural substrate, preventing its dissociation. These results elucidate a paradox which has been unexplained for more than 20 years and further validates both the proposed substrate and inhibition chemical mechanisms.     

Photochemistry of 4'-benzophenone-substituted nucleoside derivatives as models for ribonucleotide reductases: competing generation of 3'-radicals and photoenols

Ribonucleotide reductases (RNRs) catalyze the 2'-reduction of ribonucleotides, thus providing 2'-deoxyribonucleotides, the monomers for DNA-biosynthesis. The current mechanistic hypothesis for the catalysis effected by this class of enzymes involves a sequence of radical reactions. A 3'-hydrogen abstraction, effected by a radical at the enzyme's active site, is believed to initiate the catalytic cycle. As models for this substrate-enzyme interaction, the photochemically induced intramolecular hydrogen abstraction in a series of 4'-benzophenone-substituted nucleoside analogues was studied. Model compounds with hydroxy-, methoxy-, mesyloxy-groups or a cyclic carbonate in 2'- and 3'-positions were investigated. Depending on the substitution pattern, two different types of photoproducts were observed: Those which result from photoenol formation (gamma-H-abstraction) and those which result from abstraction of the 3'-H-atom (delta-H-abstraction). Photoenol formation was further supported by H/D-exchange experiments. Thus, the 3'-H-abstraction postulated as the initial step in RNR action was successfully modeled by photolysis of 4'-benzophenone-substituted nucleoside analogues. The regioselectivity of the photochemical H-abstraction and thus of the product distribution as a function of the 2'- and 3'-substituents was rationalized on the basis of a conformational analysis of the four model systems, utilizing molecular mechanics simulations.     

Direct determination of rate constants for coupling between aromatic radical anions and alkyl and benzyl radicals by laser-flash photolysis

Coupling rates between the radicals methyl, n-, sec-, tert-butyl and benzyl (R.) and the aromatic radical anions of 1,4-dicyanonaphthalene, 9,10-dicyanoanthracene and fluorenone (A-.) have been obtained using a new laser-flash photolysis method. The radicals R. and the radical anions A-. were generated by a photoinduced electron transfer reaction between the aromatic compound A and the alkyl or benzyl triphenylborate anion RB(Ph)3-. For the first time the rate constants of the coupling reaction between methyl and benzyl radicals with aromatic radical anions have been obtained. For all the measured coupling rate constants an average value of k1 = 1.9 x 10(9) M-1 s-1 was found with a relatively small variation in the coupling rates (0.8-2.9 x 10(9) M-1 s-1). The results demonstrate that the coupling rate k1 is insensitive to changes in the steric and electronic properties of the radicals and the structure and standard potentials of the aromatic radical anions.     

Oxidation of 2-deoxyribose by benzotriazinyl radicals of antitumor 3-amino-1,2,4-benzotriazine 1,4-dioxides

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead bioreductive drug in clinical trials as an anticancer agent to kill refractory hypoxic cells of solid tumors. It has long been known that, upon metabolic one-electron reduction, tirapazamine induces lethal DNA double strand breaks in hypoxic cells. These strand breaks arise from radical damage to the ribose moiety of DNA, and in this pulse radiolysis and product analysis study we examine mechanistic aspects of the dual function of tirapazamine and analogues in producing radicals of sufficient power to oxidize 2-deoxyribose to form radicals, as well as the ability of the compounds to oxidize the resulting deoxyribose radicals to generate the strand breaks. Both the rate of oxidation of 2-deoxyribose and the radical yield increase with the one-electron reduction potentials of the putative benzotriazinyl radicals formed from the benzotriazine 1,4-dioxides. Subsequent oxidation of the 2-deoxyribose radicals by the benzotriazine 1,4-dioxides and 1-oxides proceeds through adduct formation followed by breakdown to form the radical anions of both species. The yield of the radical anions increases with increasing one-electron reduction potentials of the compounds. We have previously presented evidence that oxidizing benzotriazinyl radicals are formed following one-electron reduction of the benzotriazine 1,4-dioxides. The reactions reported in this work represent the kinetic basis of a short chain reaction leading to increased oxidation of 2-deoxyribose, a process which is dependent on the one-electron reduction potential of the benzotriazinyl radicals that are above a threshold value of ca. 1.24 V.     

Formation of spectral intermediate G-C and A-T anion complex in duplex DNA studied by pulse radiolysis

The dynamics of electron adducts of 2'-deoxynucleotides and oligonucelotides (ODNs) were measured spectroscopically by nanosecond pulse radiolysis. The radical anions of the nucleotides were produced within 10 ns by the reaction of hydrated electrons (e(aq)(-)) and were protonated to form the corresponding neutral radicals. At pH 7.0, the radical anion of deoxythymidine (dT(*-)) was protonated to form the neutral radical dT(H)(*) in the time range of microseconds. The rate constant for the protonation was determined as 1.8 x 10(10) M(-1) s(-1). In contrast, the neutral radical of dC(H)(*) was formed immediately after the pulse, suggesting that the protonation occurs within 10 ns. The transient spectra of excess electrons of the double-stranded ODNs 5'-TAATTTAATAT-3' (AT) and 5'-CGGCCCGGCGC-3' (GC) differed from those of pyrimidine radicals (C and T) and their composite. In contrast, the spectra of the electron adducts of the single-stranded ODNs GC and AT exhibited characteristics of C and T, respectively. These results suggest that, in duplex ODNs, the spectral intermediates of G-C and A-T anions complex were formed. On the microsecond time scale, the subsequent changes in absorbance of the ODN AT had a first-order rate constant of 4 x 10(4) s(-1), reflecting the protonation of T.     

Investigation of reactions postulated to occur during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Model 3'-azido-3'-deoxynucleosides with thiol or vicinal dithiol substituents at C2' or C5' were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides. Esterification of 5'-(tert-butyldiphenylsilyl)-3'-azido-3'-deoxyadenosine and 3'-azido-3'-deoxythymidine (AZT) with 2,3-S-isopropylidene-2,3-dimercaptopropanoic acid or N-Boc-S-trityl-L-cysteine and deprotection gave 3'-azido-3'-deoxy-2'-O-(2,3-dimercaptopropanoyl or cysteinyl)adenosine and the 3'-azido-3'-deoxy-5'-O-(2,3-dimercaptopropanoyl or cysteinyl)thymidine analogs. Density functional calculations predicted that intramolecular reactions between generated thiyl radicals and an azido group on such model compounds would be exothermic by 33.6-41.2 kcal/mol and have low energy barriers of 10.4-13.5 kcal/mol. Reduction of the azido group occurred to give 3'-amino-3'-deoxythymidine, which was postulated to occur with thiyl radicals generated by treatment of 3'-azido-3'-deoxy-5'-O-(2,3-dimercaptopropanoyl)thymidine with 2,2'-azobis-(2-methyl-2-propionamidine) dihydrochloride. Gamma radiolysis of N(2)O-saturated aqueous solutions of AZT and cysteine produced 3'-amino-3'-deoxythymidine and thymine most likely by both radical and ionic processes.     

PELDOR spectroscopy with DOPA-beta2 and NH2Y-alpha2s: distance measurements between residues involved in the radical propagation pathway of E. coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, alpha2 and beta2. The alpha2 is the site of nucleotide reduction, and beta2 harbors a diferric tyrosyl radical (Y122*) cofactor. Turnover requires formation of a cysteinyl radical (C439*) in the active site of alpha2 at the expense of the Y122* in beta2. A docking model for the alpha2beta2 interaction and a pathway for radical transfer from beta2 to alpha2 have been proposed. This pathway contains three Ys: Y356 in beta2 and Y731/Y730 in alpha2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these alpha2/beta2 variants and PELDOR spectroscopy to measure the distance between the Y122* in one alphabeta pair and the newly formed radical in the second alphabeta pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439* generation and provide a structural constraint for residue Y356, which is thermally labile in all beta2 structures solved to date.     

PELDOR study on the tyrosyl radicals in the R2 protein of mouse ribonucleotide reductase

Pulse electron-electron double resonance (PELDOR) has been employed to measure the distance between the putative tyrosyl radicals in the two halves of the R2 subunit from mouse ribonucleotide reductase. The results provide experimental evidence that the active, tyrosyl radical containing mouse R2 subunit forms a homodimeric form in solution. The distance between the two tyrosyl radicals present in the dimer was determined to be 3.25 +/- 0.05 nm.     

Theoretical studies on the mechanism of inhibition of Ribonucleotide Reductase by (E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate

(E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate (FMCDP) is a potent time-dependent inactivator of the enzyme Ribonucleotide Reductase, which operates by destructing an essential tyrosil radical and performing a covalent addition to an active site residue. Considerable effort to elucidate the inhibition mechanism has been undertaken in recent years, and some insights have been obtained. Although a mechanistic proposal has been put forward, based on a general paradigm of inhibition of RNR by 2' substituted substrate analogues, details about the mechanism have remained elusive. Namely, the exact residue that adds to the inhibitor is still not identified, although mutagenesis experiments suggest that it should correspond to the E441 residue. In this work, we performed an extensive theoretical exploration of the potential energy surface of a model system representing the active site of RNR with FMCDP, using Density Functional Theory. This study establishes the detailed mechanism of inhibition, which is considerably different from the one proposed earlier. The proposed mechanism is fully consistent with available experimental data. Energetic results reveal unambiguously that the residue adding to the inhibitor is a cysteine, most probably C439, and exclude the possibility of the addition of E441. However, the E441 residue is shown to be essential for inhibition, catalyzing both the major and minor inhibition pathways, in agreement with mutagenesis results. It is shown also that the major mode of inactivation mimics the early stages of the natural substrate pathway.     

Anthralin: primary products of its redox reactions

One-electron reduction significantly enhances the ability of anthralin, 1, to act as a hydrogen atom donor. On annealing of an MTHF glass in which the radical anion of anthralin, 1*-, is generated radiolytically, this species decays mainly by loss of H* to give the anthralyl anion, 2- . On the other hand, radicals formed on radiolysis of matrices that are suitable for the generation of radical anions or cations are capable to abstract H* from anthralin to give the anthralyl radical, 2* . Both 2- and 2* are obtained simultaneously by mesolytic cleavage of the radical anion of the anthralin dimer. Contrary to general assumptions, the anthralyl radical is found to be much more reactive toward oxygen than the anion. All intermediates are characterized spectroscopically and by reference to quantum chemical calculations. Attempts to generate the radical cation of anthralin by X-irradiation of an Ar matrix containing anthralin led also to significant formation of its radical anion, i.e., anthralin acts apparently as an efficient electron trap in such experiments.     

One electron oxidation induced dimerization of 5-hydroxytryptophol: role of 5-hydroxy substitution

Reaction of one-electron oxidant (Br(2)(*-)) with tryptophol (TP) and 5-hydroxytryptophol (HTP) have been studied in aqueous solution in the pH range from 3 to 10, employing nanosecond pulse radiolysis technique and the transients detected by kinetic spectrophotometry. One-electron oxidation of TP has produced an indolyl radical that absorbs in the 300-600 nm region with radical pK(a) = 4.9 +/- 0.2, while the reaction with HTP has produced an indoloxyl radical with lambda(max) at 420 nm and radical pK(a) < 3. Hydroxyl radicals ((*)OH) react with these two compounds producing (*)OH radical adducts that undergo water elimination to give one-electron-oxidized indolyl and indoloxyl radical species, respectively. The indoloxyl radicals react with the parent compound to form dimer radicals with an average association constant of (6.7 +/- 0.4) x 10(4) M(-1). No such dimerization is observed with indolyl radical, indicating that the presence of the 5-hydroxy group markedly alters its ability to form a dimer. A possible explanation behind such a difference in reactivity has been supported with ab initio quantum chemical calculations.     

Do Photolyases Need To Provide Considerable Activation Energy for the Splitting of Cyclobutane Pyrimidine Dimer Radical Anions?

cis-syn Cyclobutane pyrimidine dimers, major UV-induced DNA lesions, are efficiently repaired by DNA photolyases. The key step of the repair reaction is a light-driven electron transfer from the FADH(-) cofactor to the dimer; the resulting radical anion splits spontaneously. Whether the splitting reaction requires considerable activation energy is still under dispute. Recent reports show that the splitting reaction of a dimer radical anion has a significant activation barrier (0.45 eV), and so photolyases have to provide considerable energy. However, these results contradict observations that cis-syn dimer radical anions split into monomers at -196 degrees C, and that the full process of DNA photoreactivation was fast (1.5-2 ns). To investigate the activation energies of dimer radical anions, three model compounds 1-3 were prepared. These include a covalently linked cyclobutane thymine dimer and a tryptophan residue (1) or a flavin unit (3), and the covalently linked uracil dimer and tryptophan (2). Their properties of photosensitised splitting of the dimer units by tryptophan or flavin unit were investigated over a large temperature range, -196 to 70 degrees C. The activation energies were obtained from the temperature dependency of splitting reactions for 1 and 2, 1.9 kJ mol(-1) and 0.9 kJ mol(-1) for the thymine and uracil dimer radical anions, respectively. These values are much lower than that obtained for E. coli photolyase (0.45 eV), and are surmountable at -196 degrees C. The activation energies provide support for previous observations that repair efficiencies for uracil dimers are higher than thymine dimers, both in enzymatic and model systems. The mechanisms of highly efficient enzymatic DNA repair are discussed.     

PHOTOSENSITIZATION OF PYRIMIDINE DIMER SPLITTING BY A COVALENTLY BOUND INDOLE

Abstract— Photosensitized pyrimidine dimer splitting characterizes the enzymatic process of DNA repair by the DNA photolyases. Possible pathways for the enzymatic reaction include photoinduced electron transfer to or from the dimer. To study the mechanistic photochemistry of splitting by a sensitizer representative of excited state electron donors, a compound in which an indole is covalently linked to a pyrimidine dimer has been synthesized. This compound allowed the quantitative measurement of the quantum efficiency of dimer splitting to be made without uncertainties resulting from lack of extensive preassociation of the unlinked dimer and sensitizer free in solution. Irradiation of the compound with light at wavelengths absorbed only by the indolyl group (approximately 280 nm) resulted in splitting of the attached dimer. The quantum yield of splitting of the linked system dissolved in N₂0-saturated aqueous solution was found to be 0.04 ± 0.01. The fluorescence typical of indoles was almost totally quenched by the attached dimer. A splitting mechanism in which an electron is efficiently transferred intramolecularly from photoexcited indole to ground state dimer has been formulated. The surprisingly low quantum yield of splitting has been attributed to inefficient splitting of the resulting dimer radical anion. Insights gained from this study have important mechanistic implications for the analogous reaction effected by the DNA photolyases.     

Effect of pH on one-electron oxidation chemistry of organoselenium compounds in aqueous solutions

Pulse radiolysis coupled with absorption detection has been employed to study one-electron oxidation of selenomethionine (SeM), selenocystine (SeCys), methyl selenocysteine (MeSeCys), and selenourea (SeU) in aqueous solutions. Hydroxyl radicals (*OH) in the pH range from 1 to 7 and specific one-electron oxidants Cl2*- (pH 1) and Br2*- (pH 7) have been used to carry out the oxidation reactions. The bimolecular rate constants for these reactions were reported to be in the range of 2 x 10(9) to 10 x 10(9) M(-1) s(-1). Reactions of oxidizing radicals with all these compounds produced selenium-centered radical cations. The structure and stability of the radical cation were found to depend mainly on the substituent and pH. SeM, at pH 7, produced a monomer radical cation (lambdamax approximately 380 nm), while at pH 1, a dimer radical cation was formed by the interaction between oxidized and parent SeM (lambdamax approximately 480 nm). Similarly, SeCys, at pH 7, on one-electron oxidation, produced a monomer radical cation (lambdamax approximately 460 nm), while at pH 1, the reaction produced a transient species with (lambdamax approximately 560 nm), which is also a monomer radical cation. MeSeCys on one-electron oxidation in the pH range from 1 to 7 produced monomer radical cations (lambdamax approximately 350 nm), while at pH < 0, the reaction produced dimer radical cations (lambdamax approximately 460 nm). SeU at all the pH ranges produced dimer radical cations (lambdamax approximately 410 nm). The association constants of the dimer radical cations of SeM, MeSeCys, and SeU were determined by following absorption changes at lambdamax as a function of concentration. From these studies it is concluded that formation of monomer and dimer radical cations mainly depends on the substitution, pH, and the heteroatoms like N and O. The availability of a lone pair on an N or O atom at the beta or gamma position results in monomer radical cations having intramolecular stabilization. When such a lone pair is not available, the monomer radical cation is converted into a dimer radical cation which acquires intermolecular stabilization by the other selenium atom. The pH dependency confirms the role of protonation on stabilization. The oxidation chemistry of these selenium compounds is compared with that of their sulfur analogues.     

The 2'-deoxyadenosine-5'-phosphate anion, the analogous radical, and the different hydrogen-abstracted radical anions: molecular structures and effects on DNA damage

The 2'-deoxyadenosine-5'-phosphate (5'-dAMP) anion and its related radicals have been studied by reliably calibrated theoretical approaches. This study reveals important physical characteristics of 5'-dAMP radical related processes. One-electron oxidation of the 5'-dAMP anion is found on both the phosphoryl group and the adenine base with electron detachment energies close to that of phosphate. Partial removal of electron density from the adenine fragment leads to an extended pi system which includes the amine group of the adenine. Although the radical-centered carbon increases the extent of bonding with its adjacent atoms, it usually weakens the chemical bonds between the atoms at the alpha- and beta-positions. This tendency should be important in predicting the reactivity of the sugar-based radicals. The overall stability sequence of the H-abstracted 5'-dAMP anionic radicals is consistent with the analogous results for the H-abstracted neutral radicals of the adenosine nucleoside: aliphatic radicals > aromatic radicals. The negatively charged phosphoryl group attached to atom C(5)' of the ribose does not change this energetic sequence. All the H-abstraction produced 5'-dAMP radical anions are distonic radical anions. Studies have shown that the charge-radical-separating feature of the distonic radical anions is biologically relevant. This result should be important in understanding the reactive properties of these H-abstraction-produced anion radicals.     

Nucleophilically assisted and cationic ring-opening polymerization of tin-bridged [1]ferrocenophanes

To obtain mechanistic insight, detailed studies of the intriguing "spontaneous" ambient temperature ring-opening polymerization (ROP) of tin-bridged [1]ferrocenophanes Fe(eta-C(5)H(4))(2)SnR(2) 3a (R = t-Bu) and 3b (R = Mes) in solution have been performed. The investigations explored the influence of non-nucleophilic additives such as radicals and radical traps, neutral and anionic nucleophiles, Lewis acids, protic species, and other cationic electrophiles. Significantly, two novel methodologies and mechanisms for the ROP of strained [1]ferrocenophanes are proposed based on this study. First, as the addition of amine nucleophiles such as pyridine was found to strongly accelerate the polymerization rate in solution, a new nucleophilicallyassisted ROP methodology was proposed. This operates at ambient temperature in solution even in the presence of chlorosilanes but, unlike the anionic polymerization of ferrocenophanes, does not involve cyclopentadienyl anions. Second, the addition of small quantities of the electrophilic species H(+) and Bu(3)Sn(+) was found to lead to a cationic ROP process. These studies suggest that the "spontaneous" ROP of tin-bridged [1]ferrocenophanes may be a consequence of the presence of spurious, trace quantities of Lewis basic or acidic impurities. The new ROP mechanisms reported are likely to be of general significance for the ROP of other metallocenophanes (e.g., for thermal ROP in the melt) and for other metallacycles containing group 14 elements.     

The use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models

Low temperature electron paramagnetic resonance (EPR) spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T have been used to study radicals and metal sites in proteins and small inorganic model complexes. We have studied radicals, Fe, Cu and Mn containing proteins. For S = 1/2 systems, the high frequency method can resolve the g-value anisotropy. It was used in mouse ribonucleotide reductase (RNR) to show the presence of a hydrogen bond to the tyrosyl radical oxygen. At 285 GHz the type 2 Cu(II) signal in the complex enzyme laccase is clearly resolved from the Hg(II) containing laccase peroxide adduct. For simple metal sites, the systems over S = 1/2 can be described by the spin Hamiltonian: H(S) = BgS + D[Sz2 - S(S + 1)/3 + E/D (Sx2 - Sy2)]. From the high frequency EPR the D-value can be determined directly by, (I) shifts of g(eff) for half-integer spin systems with large D-values as observed at 345 GHz on an Fe(II)-NO-EDTA complex, which is best described as S = 3/2 system with D = 11.5 cm(-1), E = 0.1 cm(-1) and gx = gy = gz = 2.0; (II) measuring the outermost signal, for systems with small D values, distant of (2S - 1) x absolute value(D) from the center of the spectrum as observed in S= 5/2 Fe(III)-EDTA. In Mn(II) substituted mouse RNR R2 protein the weakly interacting Mn(II) at X-band could be observed as decoupled Mn(II) at 285 GHz.     

Density functional calculations on dissociation reactions of radical anions of 5-fluorouracil derivatives

Fragmentation reactions upon electron attachment to 5-fluorouracil with CH2R substituents at N1 have been evaluated by means of density functional calculations. The present results show that electron attachment to R = F, HC=O or CN derivatives follows a stepwise pathway with radical anions as intermediates. For these compounds, the most stable species formed is the pi radical anion which bears an unpaired spin density at the C6=C5-C4=O pi-conjugated system of the uracil ring. Cleavage of the N1-CH2R or N1CH2-R bond of these intermediates proceeds through the mixing of the pi and sigma states by means of proper geometrical fluctuations along the reaction coordinate. No sigma radical anion could be characterised on any of these sigma basal potential surfaces. A noticeable decrease in the activation energy for the N1-CH2R bond dissociation was observed for R = H-C=O or CN. Therefore, such derivatives with unsaturated groups positioned vicinal to the N1-C1' bond are identified as targets for the development of novel radiation-activated antitumour drugs. On the other hand, the electron transfer to the compounds with R = Cl, Br is dissociative, i.e. it occurs without the mediation of radical anions. For compounds with R = halides or R = NO2, the fragmentation of the N1CH2-R bond is the preferred dissociation pathway.