Far-UV-lnduced Dimeric Photoproducts in Short Oligonucleotides: Sequence Effects

Cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone adducts represent the two major classes of far-UV-induced DNA photoproducts. Because of the lack of appropriate detection methods for each individual photoproduct, little is known about the effect of the sequence on their formation. In the present work, the photoproduct distribution obtained upon exposure of a series of dinucleoside monophosphates to 254 nm light was determined. In the latter model compounds, the presence of a cytosine, located at either the 5′- or the 3′- side of a thymine moiety, led to the preferential formation of (6-4) adducts, whereas the cis-syn cyclobutane dimer was the main thymine-thymine photoproduct. In contrast, the yield of dimeric photoproducts, and particularly of (6-4) photoadducts, was very low upon irradiation of the cytosine–cytosine dinucleoside monophosphate. However, substitution of cytosine by uracil led to an increase in the yield of (6-4) photoproduct. It was also shown that the presence of a phosphate group at the 5′- end of a thymine-thymine dinucleoside monophosphate does not modify the photoproduct distribution. As an extension of the studies on dinucleoside monophosphates, the trinucleotide TpdCpT was used as a more relevant DNA model. The yields of formation of the thymine-cytosine and cytosine–thymine (6-4) photoproducts were in a 5:1 ratio, very close to the value obtained upon photolysis of the related dinucleoside monophosphates. The characterization of the two TpdCpT (6-4) adducts was based on H NMR, UV and mass spectroscopy analyses. Additional evidence for the structures was inferred from the analysis of the enzymatic digestion products of the (6-4) adducts of TpdCpT with phosphodiesterases. The latter enzymes were shown to induce the quantitative release of the photoproduct as a modified dinucleoside monophosphate in a highly sequence-specific manner.     

Photochemical Reactions of Alkyl Phenylglyoxylates(1)

Three new photoproducts, ethyl O-benzoyl mandelate (5a), ethyl O-acetylmandelate (6a), and biphenyl triketone (7a) are isolated and identified in the reactions of ethyl phenylglyoxylate (1a) in benzene. Quantum yields and initial rate constants of product formation are shown to be concentration dependent. For the formation of carbonyl product 3 at lower starting material concentrations (<0.01 M), quantum yields greater than 1 are observed. Variations in the quantum yields as a function of reaction time are due to the accumulation of alpha-hydroxyphenyl ketene (D). The relative reactivities of triplet excited states of phenylglyoxylates 1 and phenyl ketones are compared. A mechanism involving both intramolecular gamma-H abstraction and intermolecular H abstraction, which leads to radical chain reaction, is proposed. Rate constants for intramolecular gamma-H abstraction (k(N)) and intermolecular H abstraction (k(I)) of methyl phenylglyoxylate (1d) are measured.     

PHOTOCHEMISTRY OF URACIL. INTERSYSTEM CROSSING AND DIMERIZATION IN AQUEOUS SOLUTION

Abstract— Ultraviolet irradiation of ¹⁴C-uracil in aqueous solution results in the formation of hydrate and dimer photoproducts. The rate of dimerization increases with increasing uracil concentration, and decreases with increasing concentration of oxygen in solution. The kinetics are in agreement with a model previously proposed to account for the reactions, in which dimerization occurs by a reaction involving the triplet state of uracil, but hydration occurs from an excited singlet state. Oxygen reduces dimer formation by quenching the triplet. The quantum yield for intersystem crossing (ISC) to the triplet depends on the irradiation wavelength, increasing from 0.0014 at 280 nm to 0.016 at 230 nm. The ratio of rate constants for reaction of the triplet with oxygen and for dimerization is 1.1; the ratio of rate constants for triplet decay and for dimerization is 5.9 × 10^-5 M. The increase in ISC with photon energy suggests that ISC is favoured from excited vibrational levels. The quantum yield for hydration is about 0.002 at pH 4.5 for all wavelengths, but increases as the pH is decreased.     

WAVELENGTH DEPENDENT FORMATION OF THYMINE DIMERS AND (6-4)PHOTOPRODUCTS IN DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT RANGING FROM 150 TO 365 nm

We investigated the wavelength dependence of cyclobutane thymine dimer and (6-4)photoproduct induction by monochromatic UV in the region extending from 150 to 365 nm, using an enzyme-linked immunosorbent assay with two monoclonal antibodies. Calf thymus DNA solution was irradiated with 254-365 nm monochromatic UV from a spectrograph, or with 220-300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6-4)photoproducts were fluence-dependently induced by every UV below 220 nm extending to 150 nm under dry condition. We detected the efficient formation of both types of damage in the shorter UV region, as well as at 260 nm, which had been believed to be the most efficient wavelength for the formation of UV lesions. The action spectra for the induction of thymine dimers and (6-4)photoproducts were similar from 180 to 300 nm, whereas the action spectrum values for thymine dimer induction were about 9- and 1.4-fold or more higher than the values for (6-4)photoproduct induction below 160 nm and above 313 nm, respectively.     

PHOTOCHEMICAL REACTIONS OF THYMINE, URACIL, URIDINE, CYTOSINE AND BROMOURACIL IN FROZEN SOLUTION AND IN DRIED FILMS*

Abstract— The photochemistry of uracil, uridine, cytosine, thymine and broinouracil has been investigated in frozen aqueous solution and in dried films. Essentially the same photoproducts were obtained in the two conditions; however, the yield of photoproducts was considerably greater in frozen solution. Uracil forms a dimer which can exist in two forms. Some kinetic data are presented for the interconversion of these two forms, The mixed dimer of thymine and uracil can also exist in two forms. Uridine forms only one acid stable photoproduct and does not appear to form mixed photoproducts under the conditions used. Two new photoproducts of thymine other than the dimer are described. Cytosine was at first considered to be completely inert hut using more sensitive detecting equipment it has recently been found to form uracil dinier as a result of dinierization and deamination. The most remarkable response was shown by bromouracil. When irradiated by itself it formed no photoproducts but when irradiated in the presence of uracil, uridine, cytosine or NaOH it formed many photoproducts. Most of these products were devoid of bromide, but two still contained bromine. One of these has been identified as the mixed dimer of uracil and bromouracil while the other has been tentatively identified as the dimer of bromouracil. Dimers of thymine or bromouracil were not formed by X-rays.     

Excited- and ground-state versions of the tri-pi-methane rearrangement: mechanistic and exploratory organic photochemistry

The di-pi-methane rearrangement with two pi-groups bonded to a single carbon leading to pi-substituted cyclopropanes is now well established. The present research had as its goal the exploration of molecular systems having three pi-moieties attached to an sp(3)-hybridized atom in a search for a tri-pi-methane rearrangement. Indeed, it was found that such systems do rearrange photochemically to afford cyclopentenes. However, it was also established that vinylcyclopropanes ring-expand to cyclopentenes on direct irradiation. Since both three-ring and five-ring photoproducts often are found to be produced, it was important to establish that the observed photochemistry was really the result of a true single-step tri-pi-methane rearrangement and not the consequence of two sequential rearrangements, first to form a vinyl cyclopropane which subsequently ring expanded to the cyclopentene. The general situation has three species-A, B, and C-corresponding to tri-pi-methane reactant A, vinylcyclopropane photoproduct B, and cyclopentene photoproduct C. Three rate constants are involved, k(1) for A --> B, k(2) for A --> C, and k(3) for B --> C. The kinetics were applied to two examples with provision to avoid differential light absorption; this utilized singlet sensitization. It was determined that direct formation of the cyclopentene photoproduct proceeds more rapidly than the ring-expansion route. In contrast to the di-pi-methane rearrangement, the tri-pi-methane reaction was found to be preferred by the singlet, while in these sterically congested systems, the triplet led to di-pi-methane reactivity. Finally, a ground-state counterpart of the reaction was obtained.     

Photochemistry of a naphthalene-thymine dyad in the presence of acetone

Irradiation of dyad 1 in aqueous acetone leads to the introduction of an acetonyl substituent at the naphthalene 5-position, to give photoproduct 2. The proposed reaction mechanism involves electron transfer from the naphthalene excited singlet state to the ketone. Neither thymine dimers, nor acetone photoadducts involving the thymine ring were detected. These photoproducts would arise from the thymine triplet excited state, which in dyad 1 must be efficiently depopulated via a fast intramolecular energy transfer to the naphthalene chromophore, due to the lower energy of its excited triplet state.     

Photochemical Hydrogen Abstraction and Electron Transfer Reactions of Tetrachlorobenzoquinone with Pyrimidine Nucleobases

Pentachlorophenol, a widespread environmental pollutant that is possibly carcinogenic to humans, is metabolically oxidized to tetrachloroquinone (TCBQ) which can result in DNA damage. We have investigated the photochemical reaction dynamics of TCBQ with two pyrimidine type nucleobases (thymine and uracil) upon UVA (355 nm) excitation using the technique of nanosecond time-resolved laser flash photolysis. It has been found that 355 nm excitation populates TCBQ molecules to their triplet state ³TCBQ^*, which are highly reactive towards thymine or uracil and undergo two parallel reactions, the hydrogen abstraction and electron transfer, leading to the observed photoproducts of TCBQH· and TCBQ·^- in transient absorption spectra. The concomitantly produced nucleobase radicals and radical cations are expected to induce a series of oxidative or strand cleavage damage to DNA afterwards. By characterizing the photochemical hydrogen abstraction and electron transfer reactions, our results provide potentially important molecular reaction mechanisms for understanding the carcinogenic effects of pentachlorophenol and its metabolites TCBQ.     

A theoretical characterization of the photoisomerization channels of 1,2-cyclononadienes on both singlet and triplet potential-energy surfaces

The ground-, (1)(pipi*)-, and (3)(pipi*)-state potential-energy surfaces of 1,2-cyclononadiene and isomeric C(9)H(14) species, as well as 1-methyl-1,2-cyclononadiene and isomeric C(10)H(16) species were all mapped using CASSCF and the 6-31G(d) basis set. Theoretical results were found to be in good agreement with the available experimental observations for both 1,2-cyclononadiene and 1-methyl-1,2-cyclononadiene isomerization reactions under singlet and triplet direct or sensitized irradiation. Extremely efficient decay occurs from the first singlet excited state to the ground state through at least three different conical intersections (surface crossings). The first of these crossing points is accessed by a one-bond ring closure. From this conical intersection point (CI-A or CI-C), some possible subsequent ground-state reaction paths have been identified: 1) intramolecular C--H bond insertion to form the bicyclic photoproduct and 2) intramolecular C--H bond insertion to form tricyclic photoproducts. An excited state [1,3]-sigmatropic shift leads to the second conical intersection (CI-B or CI-E), which can give a three-bond cyclononyne species. Besides these, in the singlet photochemical reactions of 1-methyl-1,2-cyclononadiene, excited-state, one allenic C--H bond insertion leads to a third conical intersection (CI-D). Possible ground-state reaction pathways from this structure lead to the formation of a diene photoproduct or to transannular insertion photoproducts. Moreover, in the case of triplet 1,2-cyclononadiene and 1-methyl-1,2-cyclononadiene photoisomerization reactions, both chemical reactions will adopt a 1,3-biradical (T(1)/S(0)-1, T(1)/S(0)-2, and T(1)/S(0)-3), which may undergo intersystem crossings leading to the formation of tricyclic or bicyclic photoproducts. The results obtained allow a number of predictions to be made.     

Coumarin 314 free radical cation: formation, properties, and reactivity toward phenolic antioxidants

We have explored the photogeneration of the coumarin 314 radical cation by using nanosecond laser excitation at wavelengths longer than 400 nm in benzene, acetonitrile, dichloromethane, and aqueous media. In addition, time-resolved absorption spectroscopy measurements allowed detection of the triplet excited state of coumarin 314 (C(314)) with a maximum absorption at 550 nm in benzene. The triplet excited state has a lifetime of 90 μs in benzene. It is readily quenched by oxygen (k(q) = 5.0 × 10(9) M(-1) s(-1)). From triplet-triplet energy transfer quenching experiments, it is shown that the energy of this triplet excited state is higher than 35 kcal/mol, in accord with the relatively large singlet oxygen quantum yield (Φ(Δ) = 0.25). However, in aqueous media, the coumarin triplet was no longer observed, and instead of that, a long-lived (160 μs in air-equilibrated solutions) free radical cation with a maximum absorbance at 370 nm was detected. The free radical cation generation, which has a quantum yield of 0.2, occurs by electron photoejection. Moreover, density functional theory (DFT) calculations indicate that at least 40% of the electronic density is placed on the nitrogen atom in aqueous media, which explains its lack of reactivity toward oxygen. On the other hand, rate constant values close to the diffusion rate limit in water (>10(9) M(-1) s(-1)) were found for the quenching of the C(314) free radical cation by phenolic antioxidants. The results have been interpreted by an electron-transfer reaction between the phenolic antioxidant and the radical cation where ion pair formation could be involved.     

Triplet-sensitized photolysis of the photoisomer of a 2,11-diaza[3,3](9,10)anthracenoparacyclophane: an adiabatic cycloreversion and a [2pia + 2pia + 2sigmas] rearrangement in a triplet state of the biplanophane system

The triplet-sensitized photoreactions of the title biplanophane system 6, the photoisomer of a 2,11-diaza[3,3](9,10)anthracenoparacyclophane derivative 5, were investigated by stationary and laser-flash photolyses using xanthone (XT) and benzophenone (BP) as triplet sensitizers. When photoisomer 6 underwent XT-sensitized irradiation, a triplet cyclophane 5 and a novel polycyclic product 7 were obtained via an adiabatic cycloreversion and a formal [2pia + 2pia + 2sigmas] rearrangement, respectively. The maximum quantum yield for the formation of cyclophane 5 (0.69) and the upper-limit efficiency for the formation of polycycle 7 (0.31) were determined by laser photolysis techniques. For BP-sensitized photolysis of photoisomer 6, oxetane 8, in addition to triplet cyclophane 5 and polycycle 7, was formed by a Paterno-Buchi reaction. The quenching rate constant (k(q)) of triplet BP by photoisomer 6 (3.4 x 10(8) dm(3) mol(-)(1) s(-)(1)) was found to be 1 order of magnitude smaller than that for XT (5.0 x 10(9) dm(3) mol(-)(1) s(-)(1)). On the basis of the relationship between k(q) and the triplet donor-acceptor energy gap, the triplet energy level of photoisomer 6 was estimated to be approximately 71 kcal mol(-)(1). The photochemical and the photophysical processes involved in the sensitized photolyses are summarized in an energetic reaction diagram and discussed in detail.     

THE PHOTOCHEMISTRY OF 14 C-5-BROMO-2''-DEOXYURIDYLYL-(3''→5'')-THYMIDINE DETERMINATION OF QUANTUM YIELDS AS A FUNCTION OF pH

Abstract— Quantum yields for the formation of the major photoproduct of 2-¹⁴C-5-bromo-2'-deoxyuridylyl-(3'→5')-thymidine (BrdUpT) have been determined by irradiation of the 2-¹⁴C-BrU-labeled dinucleotide at pH 2.3, 5.9, 7.05, 8.0 and 10.25 with U.V. light at 280nm. At acidic and neutral pH the quantum yield was 0.0063; the value decreased markedly above pH 8.0 to 0.0025 at pH 10.25. Some evidence of the formation of additional photoproducts at high and low pH was found. Some aspects of the mechanism of the reaction are discussed.
Consideration of p K_a values calculated for singlet and triplet excited states indicates that the decrease in the quantum yield of main photoproduct at high pH is due to dissociation of the excited bromodeoxyuridine moiety. It is suggested that the formation of BrdUpT photoproduct and the debromination of bromouracil-labeled DN A occur via different excited states.     

Effect of borate buffer on the photolysis of riboflavin in aqueous solution

The photolysis of riboflavin (RF) in the presence of borate buffer (0.1-0.5M) at pH 8.0-10.5 has been studied using a specific multicomponent spectrophotometric method for the determination of RF and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF). The overall first-order rate constants for the photolysis of RF (1.55-4.36 x 10(-2)min(-1)) and the rate constants for the formation of FMF (1.16-3.52 x 10(-2)min(-1)) and LC (0.24-0.84 x 10(-2)min(-1)) have been determined. The values of all these rate constants decrease with an increase in buffer concentration suggesting the inhibition of photolysis reaction by borate species. The kinetic data support the formation of a RF-borate complex involving the ribityl side chain to cause the inhibition of photolysis. The second-order rate constants for the borate inhibited reaction range from 1.17-3.94 x 10(-2)M(-1)min(-1). The log k-pH profiles for the reaction at various buffer concentrations indicate a gradual increase in rate, with pH, up to 10 followed by a decrease in rate at pH 10.5 probably due to ionization of RF and quenching of fluorescence by borate species. A graph of second-order rate constants against pH is a sigmoid curve showing that the rate of photolysis increases with an increase in pH. The results suggest the involvement of excited singlet state, in addition to excited triplet state, in the formation of LC.     

ULTRAVIOLET-INDUCED REACTIONS OF THYMINE AND URACIL IN THE PRESENCE OF CYSTEINE

Abstract —Ultraviolet-radiation photolysis of thymine in the presence of cysteine gives rise to four isomeric dimers, dihydrothymine, and at least five cysteine addition products. Similar reactions occur for uracil but the products have not all been characterized in detail. The addition reactions arise from the triplet state of the pyrimidine. The initial step is production of a hydropyrimidine radical, which then reacts with cysteine to give the addition products. The triplet is quenched by cysteine with a rate constant of about 2 times 10⁸ M^-1 s^-1 for thymine and 2–9 times 10⁸ for uracil. The total yield of products gives a lower-limit estimate of the triplet yield and hence of the intersystem-crossing efficiency. These studies, combined with earlier determinations of dimer yields, show that 93% of the thymine triplets which interact with another thymine molecule are quenched without forming stable dimers. For uracil, the corresponding figure is 75%.     

Structure determination of an interstrand-type cis-anti cyclobutane thymine dimer produced in high yield by UVB light in an oligodeoxynucleotide at acidic pH

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[ c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3'-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.     

WAVELENGTH DEPENDENCE (150–290 nm) OF THE FORMATION OF THE CYCLOBUTANE DIMER AND THE (6–4) PHOTOPRODUCT OF THYMINE

The action cross sections for the formation of the cyclobutane dimer and the (6-4) photoproduct of thymine as well as the absorption cross sections of thymine were determined in the wavelength region between 150 and 290 nm. Thymine films sublimed on glass plates were irradiated by monochromatic photons in a vacuum; the induced photoproducts were quantitatively analyzed by high-performance liquid chromatography (HPLC). Under our conditions, two major peaks appeared on the HPLC chromatograms of irradiated samples. The two peaks were identified as being the cis-syn cyclobutane dimer and the (6-4) photoproduct, based on their HPLC retention times, absorption spectra in the effluent, and photochemical reactivity. The fractions of the two photoproducts increased linearly with the fluence at low fluences over the entire wavelength range. Their action cross sections were determined by the slopes of the linear fluence response curve at 10 nm intervals between 150 and 290 nm. The two action spectra showed a similar wavelength dependence and had a maximum at 270 nm as well as two minor peaks at 180 and 220 nm, at which wavelengths the peaks of the absorption spectrum of thymine sublimed on a CaF2 crystal plate appeared. The quantum yields had relatively constant values of around 0.008 for the dimer and 0.013 for the (6-4) photoproduct above 200 nm, decreasing to 0.003 and 0.006, respectively, at 150 nm as the wavelength became shorter.     

TRIPLET EXCITED STATE OF THE 4'5' PHOTOADDUCT OF PSORALEN AND THYMINE

Abstract— The triplet-triplet absorption spectrum of the 4'5' psoralen-thymine mono-adduct has been determined in water and methanol using the technique of laser flash photolysis. The extinction coefficient of the triplet was measured by the energy-transfer method with retinol triplet as standard, and used to determine the singlet → triplet intersystem crossing quantum yield for 353 nm excitation. Reaction rate constants for mono-adduct triplet with thymine and tryptophan were measured in water. Long-lived transient absorptions detected after quenching the mono-adduct triplet with thymine and tryptophan are assigned mainly to the corresponding mono-adduct radical anion, whose spectrum was established in separate pulse radiolysis studies of the mono-adduct in aqueous formate.
The significant singlet → triplet quantum yields found for the mono-adduct might be consistent with the involvement of triplet excited mono-adduct in DNA cross-link formation, as also may be the high reactivity obtained for the triplet with thymine. The initial quenching products observed resulted from a charge-transfer reaction.     

Direct observation of a carbene-alcohol ylide

Carboethoxycarbene reacts with methanol-OD to form an ylide. The formation and decay of this ylide was monitored by ultrafast time-resolved IR spectroscopy. The formation and decay of the ylide is linearly dependent on the concentration of methanol-OD in acetonitrile with second-order rate constants of ylide formation (8.4 × 10(9) M(-1) s(-1)) and decay (1.4 × 10(9) M(-1) s(-1)). Similar results were obtained with 1-butanol.     

Ciprofloxacin photosensitized oxidation of 2'-deoxyguanosine-5'-monophosphate in neutral aqueous solution

Laser flash photolysis studies have been carried out to investigate the reactions of ciprofloxacin (CPX) with 2'-deoxyguanosine-5'-monophosphate (dGMP), N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD) and ferulic acid (FCA) in neutral aqueous solutions, respectively. CPX triplet state ((3)CPX*) can be quenched by TMPD, FCA and dGMP, with rate constants of 1.8 × 10(9), 1.5 × 10(9) and 5.8 × 10(7) dm(3) mol(-1) s(-1), respectively. TMPD radical cation (TMPD(·+)) and FCA radical cation (FCA(·+)) were observed directly. The formation rate of CPX radical anion (CPX(·-)) was determined to be 1.5 × 10(9) dm(3) mol(-1) s(-1). Redox reaction of dGMP was investigated through competing reactions using TMPD and FCA as probe. The triplet energy of CPX was determined to be 262 kJ mol(-1). Electron transfer from TMPD, FCA and dGMP to (3)CPX* was proposed.     

Ultrafast observation of a solvent dependent spin state equilibrium in CpCo(CO)

We report the observation of a solvent-dependent spin state equilibrium in the 16-electron photoproduct CpCo(CO). Time-resolved infrared spectroscopy has been used to observe the concurrent formation of two distinct solvated monocarbonyl photoproducts, both of which arise from the same triplet CpCo(CO) precursor. Experiments in different solvent environments, combined with electronic structure theory calculations, allow us to assign the two solvated photoproducts to singlet and triplet CpCo(CO)(solvent) complexes. These results add to our previous picture of triplet reactivity for 16-electron organometallic photoproducts, in which triplets were not believed to interact strongly with solvent molecules. In the case of this photoproduct, it appears that spin crossover does not present a significant barrier to reactivity, and relative thermodynamic stabilities determine the spin state of the CpCo(CO) photoproduct in solution on the picosecond time scale. While the existence of transition metal complexes with two thermally accessible spin states is well-known, this is, to our knowledge, the first observation of a transient photoproduct that exhibits an equilibrium between two stable spin states, and also the first observed case in which a solvent has been able to coordinate as a token ligand to two spin states of the same photoproduct.