Ring opening of the cyclobutane in a thymine dimer radical anion

The reactions of hydrated electrons (e(aq) (-)) with thymine dimer 2 and thymidine have been investigated by radiolytic methods coupled with product studies, and addressed computationally by means of BB1K-HMDFT calculations. Pulse radiolysis revealed that one-electron reduction of the thymine dimer 2 affords the radical anion of thymidine (5) with t(1/2)<35 ns. Indeed, the theoretical study suggests that radical anion 3, in which the spin density and charge distribution are located in both thymine rings, undergoes a fast partially ionic splitting of the cyclobutane with a half-life of a few ps. This model fits with the in vivo observation of thymine dimer repair in DNA by photolyase. gamma-Radiolysis of thymine dimer 2 demonstrates that the one-electron reduction and the subsequent cleavage of the cyclobutane ring does not proceed by means of a radical chain mechanism, that is, in this model reaction the T(-)* is unable to transfer an electron to the thymine dimer 2.     

Pulse radiolysis study of rapid electron transfer from electron adduct and reducing OH adduct of dAMP to riboflavin and flavin adenine dinucleotide (FAD)

Rapid electron transfer from electron adduct and reducing OH adduct of 2'-deoxyadenosine-5'-monophosphate acid (dAMP) to riboflavin (RF) and flavin adenine dinudeotide (FAD) was studied using the time-resolved pulse radiolysLs techniques. Both spectroscopic and kinetic analyses showed that transient absorption spectrum of electron adduct or OH adduct of dAMP formed at first, and then changed to that of radical anion of RF or FAD after several microseconds of pulse. The evidence indicated that electron transfer from electron adduct and reducing OH adduct of dAMP to RF or FAD did occur. From buildup or bleaching kinetics of radical anions of RF and FAD, the rate constants for electron transfer were determined, respectively.     

New insights into the ultrafast photophysics of oxidized and reduced FAD in solution

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (～5.4 ps), the open (extended) conformation of the monomer (～2.8 ns), and the dimer (～27 ps). In the case of the stacked conformation of the monomer, we show that intramolecular electron transfer from the adenine to the isoalloxazine ring occurs with a time constant of 5.4 ps and is followed by charge recombination on a faster time scale, namely, 390 fs. We additionally demonstrate that deprotonated reduced flavin (FADH(-)) undergoes biphotonic ionization under high excitation fluence and dissociates into a hydrated electron and the neutral semiquinone radical FADH(?).     

Oxidative Splitting of a Pyrimidine Cyclobutane Dimer:A Pulse Radiolysis Study

The oxidative splitting process of cis-syn 1,3-dimethyluracil cyclobutane dimer(DMUD) in aqueous solution was investigated using pulse radiolysis technique.The results indicated that DMUD can be splitted into 1,3-dimethyluracil(DMU) by OH radicals(OH) and Br2 radical anions(Br2^-),but not by azide radicals(N3^).The oxidative mechanisms that an H-abstracted from DMUD for OH oxidative splitting and an electron transfer from DMUD to Br2-,were suggested.Related kinetic parameters were determined.     

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING II: REACTIONS INVOLVING PYRIMIDINE RADICAL ANION INTERMEDIATES

A series of photo-CIDNP (chemically induced dynamic nuclear polarization) experiments were performed on pyrimidine monomers and dimers, using the electron-donor Nα-acetyltryptophan (AcTrp) as a photosensitizer. The CIDNP spectra give evidence for the existence of both the dimer radical anion, which is formed by electron transfer from the excited AcTrp* to the dimer, and its dissociation product, the monomer radical anion. The AcTrp spectra are completely different from those obtained with an oxidizing sensitizer like anthraquinone-2-sulfonate, because of different unpaired electron spin density distributions in pyrimidine radical anion and cation. In the spectra of the anti (1,3-dimethyluracil) dimers, polarization is detected that originates from a spin-sorting process in the dimer radical pair, pointing to a relatively long lifetime of the dimer radical anions involved. Although the dimer radical anions of the 1,1′-trimethylene-bridged pyrimidines may have a relatively long lifetime as well, their protons have only very weak hyperfine interaction, which explains why no polarization originating from the dimer radical pair is detected. In the spectra of the bridged pyrimidines, polarized dimer protons are observed as a result of spin sorting in the monomer radical pair, from which it follows that the dissociation of dimer radical anion into monomer radical anion is reversible. A study of CIDNP intensities as a function of pH shows that a pH between 3 and 4 is optimal for observing monomer polarization that originates from spin-sorting in the monomer radical pair. At higher pH the geminate recombination polarization is partly cancelled by escape polarization arising in the same product.     

CHLORANIL-PHOTOSENSITIZED MONOMERIZATION OF DIMETHYLTHYMINE CYCLOBUTANE DIMERS and EFFECT OF MAGNESIUM PERCHLORATE

The photosensitized monomerization of the cyclobutane dimers of 1,3-dimethylthymine by p-chloranil was investigated by means of steady-state irradiation and laser-flash photolysis. Quantum yields for the monomerization are 0.34 for the cis,syn dimer, 0.39 for the trans,syn dimer, and much less than 10(-2) for the cis,anti isomer. Formation of the chloranil anion radical associated with quenching of triplet chloranil by the dimers demonstrates that electron transfer from dimers to triplet chloranil occurs to initiate the monomerization. Kinetic analysis suggested that the syn-dimer cation radicals undergo the ring cleavage at greater than or equal to 10(9) s-1 before escaping from the solvent cage, while the reactivity of the anti-dimer cation radical is very low. The different reactivities of the syn and anti dimer cation radicals are discussed in terms of through-bond coupling between the n orbitals of N(1) and N(1') involving the cyclobutane-ring sigma orbitals. In the cases of the syn-dimers, the sensitizer-dimer ion-radical pairs undergo the rapid geminate recombination that works as a major energy dissipating channel responsible for the lower-than-unity quantum yields. It has been found that the presence of Mg(ClO4)2 at 0.1 M enhances approximately 1.5 times either the monomerization of the syn dimers or the formation of the chloranil anion radical. A laser-flash photolysis study shows that Mg2+ forms a complex with either the triplet or the anion radical of chloranil. The net salt effects are attributed to the retardation of the rapid geminate recombination by the participation of Mg2+ in the sensitizer-dimer ion-radical pairs.     

Dissociative electron transfer to and from pyrimidine cyclobutane dimers: an electrochemical study

Cyclic voltammetry was used to study the reduction and oxidation behaviour of several pyrimidine cyclobutane dimers mimicking UV induced lesion in DNA strands in polar solvents (N,N-dimethylformamide and acetonitrile). Both electron injection and removal to and from the dimers, respectively, lead to their cleavage and reformation of the monomeric base. The influence of stereochemistry and substitution pattern at the cyclobutane motif on the reactivity has been studied. It appears that the repair process always proceeds in a sequential fashion with initial formation of a dimer ion radical intermediate, which then undergoes ring opening by homolytic cleavage of the two C-C bonds. Standard redox potentials for the formation of both radical anion and radical cation state of the dimers were determined. Quantum calculations on simplified model compounds reveal the reason for the finding that the exergonic homolytic cleavages of the carbon-carbon bonds are endowed with sizeable activation barriers. The consequences of these mechanistic studies on the natural enzymatic repair by photolyase enzyme are discussed.     

The dimer radical anion of acetonitrile

The dimer radical anion of acetonitrile has been identified in the upper crystalline phase of acetonitrile after γ irradiation at 77°K, whereas the monomer radical anion is produced in the lower crystalline phase under the same irradiation conditions. ESR studies on these species in aligned and polycrystalline samples are summarized and possible structures for the dimer radical anion are discussed. Dimer radical anions of other organic cyanides have also been observed in γ-irradiated crystals.     

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.     

Photoinduced N-demethylation of rufloxacin and its methyl ester under aerobic conditions

Irradiation of rufloxacin (RF) under aerobic conditions gives rise to N-demethylation of the piperazinyl ring, which is enhanced in aerated D2O. Two primary processes seem to be involved in RF N-demethylation: photoionization from 1RF and singlet oxygen generation from 3RF. Both processes may lead to the same key intermediates, namely, RF*+ and superoxide radical anion; coupling of these intermediates explains N-demethylation of RF via an iminium cation. Formation of the hydrated electron by a monophotonic process (with a quantum yield of 0.09) is detected along with 3RF (with a intersystem-crossing quantum yield phiISC = 0.36) by laser flash photolysis. Studies performed on RF methyl ester give qualitatively similar results.     

Electron attachment to a hydrated DNA duplex: the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine

The minimal essential section of DNA helices, the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine dimer octahydrate, [dGpdC](2), has been constructed, fully optimized, and analyzed by using quantum chemical methods at the B3LYP/6-31+G(d,p) level of theory. Study of the electrons attached to [dGpdC](2) reveals that DNA double strands are capable of capturing low-energy electrons and forming electronically stable radical anions. The relatively large vertical electron affinity (VEA) predicted for [dGpdC](2) (0.38 eV) indicates that the cytosine bases are good electron captors in DNA double strands. The structure, charge distribution, and molecular orbital analysis for the fully optimized radical anion [dGpdC](2)(·-) suggest that the extra electron tends to be redistributed to one of the cytosine base moieties, in an electronically stable structure (with adiabatic electron affinity (AEA) 1.14 eV and vertical detachment energy (VDE) 2.20 eV). The structural features of the optimized radical anion [dGpdC](2)(·-) also suggest the probability of interstrand proton transfer. The interstrand proton transfer leads to a distonic radical anion [d(G-H)pdC:d(C+H)pdG](·-), which contains one deprotonated guanine anion and one protonated cytosine radical. This distonic radical anion is predicted to be more stable than [dGpdC](2)(·-). Therefore, experimental evidence for electron attachment to the DNA double helices should be related to [d(G-H)pdC:d(C+H)pdG](·-) complexes, for which the VDE might be as high as 2.7 eV (in dry conditions) to 3.3 eV (in fully hydrated conditions). Effects of the polarizable medium have been found to be important for increasing the electron capture ability of the dGpdC dimer. The ultimate AEA value for cytosine in DNA duplexes is predicted to be 2.03 eV in aqueous solution.     

Primary processes in the photosensitized redox reaction of dimers of thiacarbocyanine dyes

The kinetics and the mechanism of the reaction of donor (ascorbic acid) oxidation by electron acceptors (methylviologen and p-nitroacetophenone) photosensitized by dimers of sulfoalkyl-9-ethylthiacarbocyanine dyes (Dye1, Dye2, and Dye3) were studied in aqueous solutions. Dimers of the dyes (dianions) are capable of transition to the triplet state that is mainly quenched by acceptors to form radical anions of dimers, which are unstable and dissociate within 10–12 μs into the monomer (anion) and its radical (the limiting reaction stage). The presence of a donor in the dye-acceptor mixture leads to one-electron reduction of the monomer radical to its anion followed by the dimerization reaction. The results of the analysis of the experimental data obtained by the laser photolysis technique are in good agreement with the calculated kinetic curves for the formation and the decay of the dimer radical anions.     

A comparative repair study of thymine- and uracil-photodimers with model compounds and a photolyase repair enzyme

Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed.     

Specificity of the redox photoreaction of dimers of thiadicarbocyanine dyes

The primary steps of the redox reaction of dimers of the thiadicarbocyanine dye and its 5,5′-dichloro derivative in aqueous solutions were studied in the presence of 4-nitroacetophenone, ascorbic acid, or hydroquinone. In water the dye molecules (anion, M^?) mainly exist as dimers M₂ ^2?. The laser pulse irradiation (10 ns, 532 nm) results in the population of the lowest triplet level M₂ ^2?, whose depletion occurs due to both intersystem crossing to the ground state and photoinduced transition to the highest triplet state of the dimer followed by photoionization. Photoionization at low intensities of a laser pulse proceeds via the one-quantum mechanism going to the two-quantum mechanism with an increase in the laser pulse intensity. The photooxidation of the dimer in the lowest triplet state with 4-nitroacetophenone results in the formation of unstable radical anion M₂ ^?· that spontaneously dissociates to monomer M^? and radical M^· of the dye. In the presence of electron donors (ascorbic acid, hydroquinone), the dimers in the triplet state are not photoreduced, but the electron donors reduce M₂ ^?· and M^· to the dye dimer and monomer, respectively.     

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase

Reduced anionic flavin adenine dinucleotide (FADH^?) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV‐damaged DNA. The initial step involves photoinduced electron transfer from *FADH^? to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD‐dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (k_ET) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano‐ or etheno‐bridged Ade between the AN1 and AN6 atoms (e‐FAD and ε‐FAD, respectively) were used to reconstitute apo‐PL, giving e‐PL and ε‐PL respectively. The reconstitution yield of e‐PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε‐PL showed 50% reconstitution yield. The substrate binding constants for ε‐PL and rPL were identical. ε‐PL showed a 15% higher steady‐state repair yield compared to FAD‐reconstituted photolyase (rPL). The acceleration of repair in ε‐PL is discussed in terms of an ε‐Ade radical intermediate vs superexchange mechanism.     

PHOTO-INDUCED ELECTRON EJECTION FROM THE REDUCED COPPER OF Pseudomonas aeruginosa AZURIN

The reduced form of Pseudomonas aeruginosa azurin exhibits an enhanced absorbance in the UV compared to that of the oxidized protein. This enhancement has also been observed for azurins from other bacterial species and for another type I copper protein, plastocyanin. Pulsed laser excitation of the reduced azurin in the region of enhanced absorbance at 308 nm results in single photon, rapid (less than 30 ns) oxidation of the copper center and formation of the hydrated electron with a quantum yield of 0.05. The hydrated electron reacts in the expected manner with scavengers such as nitrous oxide, oxygen, acetone and nitromethane. In the absence of scavengers, the electron reacts with the protein, including the disulfide bond, to form the disulfide radical anion, observed at 410 nm. The overall photophysical event involves a charge-transfer to solvent transition although the existence of intermediate states can not be excluded.     

Pulse radiolysis of 2-[(methacryloyloxy)ethyl]trimethylammonium chloride in aqueous solution

The early stages of the polymerization of 2-[(methacryloyloxy)ethyl]trimethyl ammonium chloride (MADQUAT) in aqueous solution were studied by pulse radiolysis. The rate constant of the reaction of this monomer with OH√ and hydrated electron was measured as 1.5×10¹⁰ and 2.2×10¹⁰ dm³mol⁻¹s⁻¹, respectively. The absorption spectrum of the OH√ adduct to MADQUAT molecule appeared to have a maximum at 290 nm. Its decay depends on the monomer concentration. The shape and maximum of the absorption spectrum of electron adduct to the monomer depend on pH of the solution. In acidic solution, radical anion is formed. It subsequently undergoes reversible protonation giving spectrum with a maximum below 260 nm. In basic solution the spectrum has a maximum at 290 nm. In this case irreversible protonation takes place at the β-carbon atom, giving an -centred carbon radical.     

Microhydration shell structure in Cl2*-.nH2O clusters: A theoretical study

We present the results of a detailed study on structure and electronic properties of hydrated cluster Cl2*-.nH2O (n = 1-7) based on a nonlocal density functional, namely, Becke's [J. Chem. Phys. 98, 1372 (1993)] half and half hybrid exchange-correlation functional with a split valence 6-311++G(d,p) basis function. Geometry optimizations for all the clusters are carried out with various possible initial guess structures without any symmetry restriction. Several minimum energy structures (conformers) are predicted with a small difference in total energy. There is a competition between the binding of solvent H2O units with Cl2*- dimer radical anion directly through ion-molecule interaction and forming interwater hydrogen-bonding network in Cl2*-.nH2O (n > or = 2) hydrated cluster. Structure having interwater H-bonded network is more stable over the structure where H2O units are connected to the solute dimer radical anion Cl2*- rather independently either by single or double H bonding in a particular size (n) of hydrated cluster Cl2*-.nH2O. At the maximum four solvent H2O units reside in interwater H-bonding network present in these hydrated clusters. It is observed that up to six H2O units are independently linked to the anion having four double H bondings and two single H bondings suggesting the primary hydration number of Cl2*- to be 6. In all these clusters, the odd electron is found to be mostly localized over the two Cl atoms and these two atoms are bound by a three-electron hemibond. Calculated interaction (between solute and different water clusters) and vertical detachment energy profiles show saturation at n = 6 in the hydrated cluster Cl2*-.nH2O (n = 1-7). However, calculated solvation energy increases with the increase in number of solvent H2O molecules in the cluster. Interaction energy varies linearly with vertical detachment energy for the hydrated clusters Cl2*-.nH2O (n < or = 6). Calculation of the vibration frequencies show that the formation of Cl2*(-)-water clusters induces significant shifts from the normal stretching modes of isolated water. A clear difference in the pattern of IR spectra is observed in the O-H stretching region of water from hexa- to heptahydrated cluster.     

Photoreactivity of biologically active compounds. XVI. Formation and reactivity of free radicals in mefloquine

The formation and reactivity of the triplet state and free radicals of mefloquine hydrochloride (MQ) have been investigated by pulse radiolysis and flash photolysis. The excited triplet, cation radical and anion radical have been produced and their absorption characteristics determined. The triplet-triplet absorption spectrum of MQ showed a maximum at 430 nm, with a molar absorption coefficient of 3600 M(-1) cm(-1) and the quantum yield for intersystem crossing was determined to be close to unity. Deactivation of the triplet, in the absence of oxygen, led to the formation of MQ cation and/or anion radicals. The molar absorption coefficient of the cation radical at 330 nm was determined to be 2300 M(-1) cm(-1), whilst that for the anion radical was 2400 M(-1) cm(-1) at 620 nm and 3600 M(-1) cm(-1) at 350 nm. The molar absorption coefficients of the proposed neutral radical at 320 nm and 520 nm were 4000 M(-1) cm(-1) and 1300 M(-1) cm(-1) respectively. The quantum yield for the formation of singlet oxygen, sensitized by MQ triplet, was determined to be close to unity. Aqueous solutions of MQ were found to photoionize to yield hydrated electron and cation radical of MQ in a biphotonic process. The influences of pH, buffer concentration, oxygen concentration and addition of sodium azide on the formation and reactivity of the transients were evaluated. The reactions between MQ and solvated electrons and superoxide anion were also studied.