Dewar Valence Isomers Research Articles

Data Processing for Predicting DNA Damaging Properties of Complex UV Sources.

A growing number of experimental evidence emphasizes that photobiological phenomena are not always the sum of the effect of individual wavelengths present in the emission spectrum of light sources. Unfortunately, tools are missing to identify such non-additive effects and predict effects of various exposure conditions. In the present work, we addressed these points for the formation of pyrimidine dimers in DNA upon co-exposure to UVC, UVB and UVA radiation. We first applied a combination index approach to determine whether mixtures of theses UV ranges exhibited additive, inhibitory or synergistic effects on the formation of cyclobutane pyrimidine dimers, (6-4) photoproducts and Dewar valence isomers. A predictive approach based on an experimental design strategy was then used to quantify the contribution of each wavelength range to the formation of DNA photoproducts. The obtained models allowed us to accurately predict the level of pyrimidine dimers in DNA irradiated under different conditions. The data were found to be more accurate than those obtained with the simple additive approach underlying the use of action spectra. Experimental design thus appears as an attractive concept that could be widely applied in photobiology even for cellular experiments.

Seasonal Differences in the UVA/UVB Ratio of Natural Sunlight Influence the Efficiency of the Photoisomerization of (6-4) Photoproducts into their Dewar Valence Isomers.

The UVA and UVB components of sunlight can produce three classes of bipyrimidine DNA photolesions [cyclobutane pyrimidine dimers (CPDs), pyrimidine (6‐4) pyrimidone photoproducts (6‐4PPs) and related Dewar valence isomers (DewarPPs)]. The UVA/UVB ratio of sunlight is high in winter and low in summer in the Northern Hemisphere. Since UVB radiation produces 6‐4PPs and UVA radiation converts them into DewarPPs through photoisomerization, it is expected that there may be differences in the photoisomerization of 6‐4PPs between summer and winter, although that has never been documented. To determine that, isolated DNA was exposed to natural sunlight for 8 h in late summer and in winter, and absolute levels of the three classes of photolesions were quantified using calibrated ELISAs. It was found that sunlight produces CPDs and 6‐4PPs in DNA at a ratio of about 9:1 and converts approximately 80% of 6‐4PPs into DewarPPs within 3 h. Moreover, photoisomerization is more efficient in winter than in late summer after sunlight irradiation for the same duration, at similar solar UV doses and with the same induction level of CPDs. These results demonstrate that seasonal differences in the UVA/UVB ratio influence the efficiency of the photoisomerization of 6‐4PPs into DewarPPs.

Pyrimidine (6‐4) Pyrimidone Photoproducts in UVA‐Irradiated DNA: Photosensitization or Photoisomerization?

AbstractFormation of pyrimidine dimers in DNA is a major initiating event in the induction of skin cancer. Model experiments suggest that, upon absorption of UVA, one type of dimers induced by UVB, the pyrimidine (6‐4) pyrimidone photoproducts, photosensitizes the formation of mutagenic cyclobutane pyrimidine dimers by triplet ‐triplet energy transfer (TTET). We investigated whether this photoreaction actually took place when 64PP were located within a DNA duplex rather than added as external sensitizers like in available data. Our results show that this process is not detectable in DNA and double‐stranded oligonucleotides exposed to a combination of UVB and UVA. TTET could only be observed, as a very minor photoreaction, in a short single‐stranded oligonucleotide bearing a 64PP. It may be concluded that 64PP‐mediated TTET does not significantly contribute to UV‐induced DNA damage. In contrast, the photoisomerization of 64PP into their Dewar valence isomers is very efficient.

The effect of DNA backbone on the triplet mechanism of UV-induced thymine-thymine (6-4) dimer formation.

Density functional theory calculations were carried out to investigate the formation mechanism of the thymine-thymine (6-4) dimer ((6-4)TT), which is one of the main DNA lesions induced by ultraviolet radiation and is closely related to skin cancers. The DNA backbone was found to have nonnegligible effects on the triplet reaction pathway, particularly the reaction steps involving substantial base rotations. The mechanism for the isomerization from (6-4)TT to its Dewar valence isomer (DewarTT) was also explored, confirming the necessity of absorbing a second photon. In addition, the solvation effects were examined and showed considerable influence on the potential energy surface. Graphical Abstract DFT calculations on the influence of DNA backbone on the mechanism of UV-induced thymine-thymine (6-4) dimer formation.

The Dewar Isomer of 1,2‐Dihydro‐1,2‐azaborinines: Isolation, Fragmentation, and Energy Storage

AbstractThe photochemistry of 1,2‐dihydro‐1,2‐azaborinine derivatives was studied under matrix isolation conditions and in solution. Photoisomerization occurs exclusively to the Dewar valence isomers upon irradiation with UV light (>280 nm) with high quantum yield (46 %). Further photolysis with UV light (254 nm) results in the formation of cyclobutadiene and an iminoborane derivative. The thermal electrocyclic ring‐opening reaction of the Dewar valence isomer back to the 1,2‐dihydro‐1‐tert‐butyldimethylsilyl‐2‐mesityl‐1,2‐azaborinine has an activation barrier of (27.0±1.2) kcal mol−1. In the presence of the Wilkinson catalyst, the ring opening occurs rapidly and exothermically (ΔH=(−48±1) kcal mol−1) at room temperature.

The Dewar Isomer of 1,2-Dihydro-1,2-azaborinines: Isolation, Fragmentation, and Energy Storage.

The photochemistry of 1,2-dihydro-1,2-azaborinine derivatives was studied under matrix isolation conditions and in solution. Photoisomerization occurs exclusively to the Dewar valence isomers upon irradiation with UV light (>280 nm) with high quantum yield (46 %). Further photolysis with UV light (254 nm) results in the formation of cyclobutadiene and an iminoborane derivative. The thermal electrocyclic ring-opening reaction of the Dewar valence isomer back to the 1,2-dihydro-1-tert-butyldimethylsilyl-2-mesityl-1,2-azaborinine has an activation barrier of (27.0±1.2) kcal mol-1 . In the presence of the Wilkinson catalyst, the ring opening occurs rapidly and exothermically (ΔH=(-48±1) kcal mol-1 ) at room temperature.

Relative Contributions of UVB and UVA to the Photoconversion of (6-4) Photoproducts into their Dewar Valence Isomers.

Dewar valence isomers are photoisomerization products of pyrimidine (6-4) pyrimidone photoproducts, a major class of UV-induced DNA lesions, which exhibits a maximal absorption around 320nm. However, Dewar isomers are not produced in significant amounts in cells exposed to biologically relevant doses of UVB. In contrast, they are readily produced when cells are exposed to a combination of UVA and UVB. The present computational work demonstrates that, on the basis of known absorption properties and formation quantum yields, the difference in Dewar formation between the two types of radiation can be explained by the role of normal bases. In the UVB range, at the low level of (6-4) photoproducts present in cells exposed to realistic doses, normal bases are present in overwhelming amounts and absorb the vast majority of the incident photons. In contrast, the absorption of DNA bases is much weaker in the UVA range while that of (6-4) photoproducts is still significant, making photoisomerization possible. This two-photon process makes it difficult to define an action spectrum for the formation of Dewar isomers.

Dewar valence isomers, the third type of environmentally relevant DNA photoproducts induced by solar radiation.

UV-induced DNA damage is the main initiating event in solar carcinogenesis. UV radiation is known to induce pyrimidine dimers in DNA, including cyclobutane dimers and (6-4) photoproducts which have been extensively studied. In contrast, much less attention has been paid to Dewar valence isomers, the photoisomerisation product of (6-4) photoproducts. Yet, the available data show that Dewar isomers can be produced by exposure to sunlight and may lead to mutations. Dewars are thus environmentally and biologically relevant. The present review summarizes currently available information on the formation, mutagenic properties and repair of this class of UV-induced DNA damage.

Pyrimidinone: versatile Trojan horse in DNA photodamage?

(6-4) Photolesions between adjacent pyrimidine DNA bases are prone to secondary photochemistry. It has been shown that singlet excited (6-4) moieties form Dewar valence isomers as well as triplet excitations. We here report on the triplet state of a minimal model for the (6-4) photolesion, 1-methyl-2(1H)-pyrimidinone. Emphasis is laid on its ability to abstract hydrogen atoms from alcohols and carbohydrates. Steady-state and time-resolved experiments consistently yield bimolecular rate constants of ∼10(4) M(-1) s(-1) for the hydrogen abstraction. The process also occurs intramolecularly as experiments on zebularine (1-(β-d-ribofuranosyl)-2(1H)-pyrimidinone) show.

Early events of DNA photodamage.

Ultraviolet (UV) radiation is a leading external hazard to the integrity of DNA. Exposure to UV radiation triggers a cascade of chemical reactions, and many molecular products (photolesions) have been isolated that are potentially dangerous for the cellular system. The early steps that take place after UV absorption by DNA have been studied by ultrafast spectroscopy. The review focuses on the evolution of excited electronic states, the formation of photolesions, and processes suppressing their formation. Emphasis is placed on lesions involving two thymine bases, such as the cyclobutane pyrimidine dimer, the (6-4) lesion, and its Dewar valence isomer.

DNA Photochemistry: Geometrically Unconstrained Pyrimidine (6–4) Pyrimidone Photoproducts Do Photoisomerize

Structural features are of major importance for the formation of mutagenic photoproducts in DNA. It was recently reported that lack of constraints between two adjacent nucleosidic units prevents the conversion of pyrimidine (6-4) pyrimidone photoproducts into their Dewar valence isomers. We here report that this is not the case for the thymidine photoproducts which, although unconstrained, are quantitatively converted into photolysis products identified as Dewar valence isomers by mass spectrometry and NMR and infrared spectroscopies.

N4-methylation of cytosine drastically favors the formation of (6-4) photoproducts in a TCG context.

Methylation of cytosine is a common biological process both in prokaryotic and eukaryotic cells. In addition to 5-methylcytosine (5mC), some bacterial species contain in their genome N(4) -methylcytosine (N4mC). Methylation at C5 has been shown to enhance the formation of pyrimidine dimeric photoproducts but nothing is known of the effect of N4 methylation on UV-induced DNA damage. In the present work, we compared the yield and the nature of bipyrimidine photoproducts induced in a series of trinucleotides exhibiting a TXG sequence where X is either T, C, 5mC or N4mC. HPLC associated to tandem mass spectrometry was used to quantify cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts (64PP) and their Dewar valence isomer. Methylation at position N4 was found to drastically increase the reactivity of C upon exposure to both UVC and UVB and to favor the formation of 64PP. In contrast methylation at C5 increased the yield of CPD at the expense of 64PP. In addition, enhancement of photoreactivity by C5 methylation was much higher in the UVB than in the UVC range. These results show the drastic effect of the methylation site on the photochemistry of cytosine.

The significance of the Dewar valence photoisomer as a UV radiation‐induced DNA photoproduct in marine microbial communities

Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by high-performance liquid chromatography-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER.

Strong impact of the solvent on the photokinetics of a 2(1H)-pyrimidinone

Pyrimidinones are part of the (6-4) photolesions which may be formed from two pyrimidine bases adjacent on a DNA strand. In relation to the secondary photochemistry of the (6-4) lesion, i.e. its transformation into a Dewar valence isomer, photophysical and photochemical properties of 1-methyl-2(1H)-pyrimidinone (1MP) in water, acetonitrile, methanol, and 1,4-dioxane are reported here. As deduced from steady state fluorescence and femtosecond transient absorption spectroscopy the S1 lifetime of 1MP is strongly affected by the solvent. The lifetimes range from 400 ps for water to 40 ps for 1,4-dioxane. Internal conversion (IC) and intersystem crossing (ISC) contribute to the S1 decay. The solvent effect on the IC rate constant is more pronounced than on the ISC constant. The quantum yields for the consumption of 1MP (values for nitrogen purged solvents) are large for methanol (0.35) and 1,4-dioxane (0.24) and small for acetonitrile (0.02) and water (0.003). Hydrogen abstraction from the solvent by the triplet state of 1MP may rationalize this.

The variety of UV-induced pyrimidine dimeric photoproducts in DNA as shown by chromatographic quantification methods

Induction of DNA damage is one of the major consequences of exposure to solar UV radiation in living organisms. UV-induced DNA photoproducts are mostly pyrimidine dimers, including cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts and Dewar valence isomers. In the last few decades, a large number of methods have been developed for the quantification of these pyrimidine dimers. The present review emphasizes the contribution of chromatographic techniques to our better understanding of the basic DNA photochemistry and the better description of damage in cells.

Strand breakage of a (6–4) photoproduct-containing DNA at neutral pH and its repair by the ERCC1–XPF protein complex

The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5' base and subsequent hydrolysis of the glycosidic bond of the 3' component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N'-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5' base was intact, whereas the glycosidic bond at the 3' component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10(-8) s(-1) at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5' fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3' end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A.

Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions†

The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.

Mechanism of the alkali degradation of (6–4) photoproduct-containing DNA

The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.

ChemInform Abstract: Chemical Investigation of Light Induced DNA Bipyrimidine Damage and Repair

AbstractReview: 55 refs.

The (5‐4) and (6‐4) Adducts of 1‐Methylthymine and Their Dewar Valence Isomers

Previous studies of the photochemical reaction of 1-methylthymine (MeT) in frozen aqueous solution have indicated that four cyclobutane type dimers are formed. We have restudied this system and have found that, in addition to cyclobutane dimers, both a (5-4) adduct and a (6-4) adduct of MeT are formed in significant amounts. Upon standing in aqueous solution, the (5-4) adduct is susceptible to reaction to form an isomeric form of the parent adduct, possibly via ring-opening and closure reactions at C-6 of the saturated pyrimidine ring component of the adduct. Irradiation of each of these three adducts with UVB light produces a pair of Dewar-type adducts. The nine products were individually characterized by mass spectrometry, proton NMR spectroscopy and UV spectroscopy. A less comprehensive study showed that irradiation of thymidine in frozen aqueous solution produces a diastereomeric pair of (5-4) adducts, along with the previously known diastereomeric pair of (6-4) adducts.