PHOTOREACTIONS AT 3655Å BETWEEN PYRIMIDINE BASES AND SKIN-PHOTOSENSITIZING FUROCOUMARINS

Abstract After irradiation at 3655 Å of an aqueous frozen solution containing thymine and psoralen, a new photocompound was isolated by column chromatography. It contains a furocoumarin and a pyrimidine-moiety linked together by the formation of a cyclobutane ring (see formulas II and III). By irradiation at 2537 Å in acetic acid solution, the photocompound breaks up again yielding psoralen and thymine. From an aqueous frozen solution containing cytosine and psoralen irradiated at 3655 Å, an analogous photocompound was obtained, which, however, consists of the addition to psoralen of a uracil molecule, instead of a cytosine one (IV, V). It has been stated that the hydrolytic deamination of the cytosine moiety to the uracil one takes place during the working up of the photocompound in aqueous solution after irradiation. Substances with properties similar to those above were obtained from bergapten (5-methoxy-psoralen) and thymine, from psoralen and thymidine or thymidylic acid, irradiated at 3655 Å.
The new substances may be considered as model compounds in explaining the photoreactions which take place between the skin-photosensitizing furocoumarins and DNA upon irradiation at 3655 Å.     

PHOTOCHEMICAL REACTIONS OF THYMINE WITH CARBOXYLIC ACIDS. A MODEL FOR NUCLEIC ACID-PROTEIN PHOTOCROSSLINKING

When nucleic acid bases are UV-irradiated in the presence of carboxylic acids or carboxylate anions new photoproducts are formed as compared to the bases irradiated in the absence of carboxylic acids. The behavior of thymine and thymidine has been examined in detail. At least four photoproducts are formed in the presence of propionic acid and three in the presence of butyric acid. None of them appears to be a cyclobutyl dimer. From the concentration dependence of the rate of photoproduct formation it is concluded that the reactive excited species is the first excited singlet state of thymine. When ¹⁴C-labelled thymine is irradiated in the presence of polyglutamic acid an important part of the radioactive material is covalently linked to the polymer. Photochemical reaction of thymine with glutamic (or aspartic) acid could thus induce crosslinks between proteins and nucleic acids. It is also shown that these photoproducts are stable under usual conditions of acidic hydrolysis of UV-irradiated DNA.     

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.     

The photochemistry of uracil: a reinvestigation

Results from a re-examination of the photochemical reactions undergone by uracil (Ura) are presented. Irradiation of Ura in frozen aqueous solution at -78.5°C produces two diastereomeric (6-4) products, namely the cis and trans isomers of 5-hydroxy-6-4'-(pyrimidin-2'-one)-5,6-dihydrouracil. Upon heating in 0.1m HCl, each of these compounds decomposes to form 6-4'-(pyrimidin-2'-one)uracil. In addition, evidence for production of a hydrate of a trimer of Ura is presented, Irradiation of this compound at 254nm forms Ura and a (6-4) adduct as products. The compounds 5-4'-(pyrimidin-2'-one)uracil and 6-hydroxy-5,6-dihydrouracil were also present after Ura was irradiated in frozen aqueous solution. Cyclobutane dimers (CBDs) are formed when Ura is irradiated in the frozen aqueous state, in fluid aqueous and acetonitrile solution and in the presence of photosensitizers (e.g. acetone). Published information, concerning the identity and relative quantitative importance of the four CBD isomers (cis-syn, cis-anti, trans-syn and trans-anti) formed in these photochemical systems, is incomplete and often in substantial disagreement. Using chemical methods in conjunction with HPLC, the identity and relative amounts of the four dimers have been determined in each of these systems. Consequently, a number of inconsistencies found in the literature concerning dimer product identity and quantitative distribution have been resolved.     

Thin‐Film Properties of DNA and RNA Bases: A Combined Experimental and Theoretical Study

The structures of the DNA and RNA bases cytosine, uracil, and thymine in thin films with a nominal film thickness of about 20 nm are studied by using X‐ray photoemission spectroscopy (XPS) and Fourier‐transform infrared spectroscopy. The molecules are evaporated in situ from powder on a gold foil. The experimental results indicate that cytosine is composed of two energetically close tautomeric forms, whereas uracil and thymine exist in only one tautomeric form. Additionally, quantum chemical calculations are performed to complement the experimental results. The relative energies of the tautomeric forms of cytosine, uracil, and thymine are calculated using Hartree–Fock (HF), density functional theory (DFT), and post‐HF methods. Furthermore, the assignment of the XPS spectra is supported by using simple model considerations employing Koopmans ionization energies and Mulliken net atomic charges.     

Investigation of proton transport tautomerism in clusters of protonated nucleic acid bases (cytosine, uracil, thymine, and adenine) and ammonia by high-pressure mass spectrometry and ab initio calculations

The energetics of the ion-molecule interactions and structures of the clusters formed between protonated nucleic acid bases (cytosine, uracil, thymine, and adenine) and ammonia have been studied by pulsed ionization high-pressure mass spectrometry (HPMS) and ab initio calculations. For protonated cytosine, uracil, thymine, and adenine with ammonia, the measured enthalpies of association with ammonia are -21.7, -27.9, -22.1, and -17.5 kcal mol-1, respectively. Different isomers of the neutral and protonated nucleic acid bases as well as their clusters with ammonia have been investigated at the B3LYP/6-31+G(d,p) level of theory, and the corresponding binding energetics have also been obtained. The potential energy surfaces for proton transfer and interconversion of the clusters of protonated thymine and uracil with ammonia have been constructed. For cytosine, the experimental binding energy is in agreement with the computed binding energy for the most stable isomer, CN01-01, which is derived from the enol form of protonated cytosine, CH01, and ammonia. Although adenine has a proton affinity similar to that of cytosine, the binding energy of protonated adenine to ammonia is much lower than that for protonated cytosine. This is shown to be due to the differing types of hydrogen bonds being formed. Similarly, although uracil and thymine have similar structures and proton affinities, the binding energies between the protonated species and ammonia are different. Strikingly, the addition of a single methyl group, in going from uracil to thymine, results in a significant structural change for the most stable isomers, UN01-01 and TN03-01, respectively. This then leads to the difference in their measured binding energies with ammonia. Because thymine is found only in DNA while uracil is found in RNA, this provides some potential insight into the difference between uracil and thymine, especially their interactions with other molecules.     

KINETICS OF DIMER FORMATION AND PHOTOHYDRATION IN ULTRAVIOLET-IRRADIATED POLYURIDYLIC ACID

Abstract— The decrease in absorbance at 2600 Å of poly-U following u.v. irradiation is analyzed quantitatively in terms of uracil-dimer formation and photohydration of uracil residues. At all wave lengths tested between 2300 and 2800 Å both dirner formation and hydration occur as well as dimer breakage. The cross section for uracil dirner breakage is large at 2300 and decreases to a relatively small value at 2800 Å. Consequently, at the shorter wave lengths the steady state for dimer formation and breakage is reached at lower doses than for the longer wave lengths. Absorbance decreases caused by longer wave lengths can be partially restored by irradiation with 2380 Å. At high doses, for all wave lengths, the main photoproducts are hydrated uracil residues. The maximum number of dimers that may be formed increases with wavelength. The absorbance loss caused by photohydration may be reversed by acidification and heat. For 2650 Å irradiation, 95 per cent of the absorbance is restored by a combination of the 2380 Å and thermal treatment. The values for the photochemical reactions of poly-U are fairly close to those for poly-T and for uridine.     

PHOTODIMERIZATION OF THYMINE AND URACIL ON FILTER PAPER*

Abstract— Thymine and uracil dimerized by ultraviolet irradiation (principally at 253 7 mμ) on filter paper and on quartz fiber sheet show a fairly good yield. These irradiation products were identical with the photodimers prepared from frozen aqueous solution. Thymine and uracil dimer in an aqueous solution reverted to the original pyrimidines by the ultraviolet irradiation with quantum yields of 0.65 and 0.50 respectively. Uracil dimer was rather labile towards hot alkaline solution. In neutral and acidic media, both dimers were quite stable. Photodimers of cytosine and isocytosine were not obtained.     

Studies on photophysics and photochemistry of N-salicylidene-p-(N,N-dimethylamino)aniline

The fluorescence spectra of N-salicylidene-p-(N,N-dimethylamino)aniline have been investigated in various solvents and three kinds of fluorescence were found; they were that of excited intermediate, exciplex and excited dimer. According to the transient absorption spectra and decay kinetic data of photoproducts of the title compound, it has been found that the photoproducts in cyclohexane are a zwitterion and a mixed dimer formed by a zwitterion and an enol; in acetonitrile the photoproducts are a zwitterion, a mixed dimer formed by a zwitterion and an enol and a dimer formed of zwitterion. Photochromic and luminescence mechanisms of the title compound are discussed as well.     

PHOTOCYCLOADDITION OF ACETONE TO URACIL AND CYTOSINE

Abstract— Ultraviolet irradiation (Λ > 280 nm) of uracil in aqueous acetone (1:1) produces cyclobutane uracil dimer and uracil-acetone addition product. The addition product is identified as an oxetane. The same product is also obtained from cytosine irradiated under the same conditions. The cytosine-acetone oxetane apparently undergoes deamination readily. Irradiation (Λ= 265 nm) of the oxetane in aqueous solution produces acetone and uracil with a quantum efficiency of 0–16.     

KINETICS OF THE ULTRAVIOLET-INDUCED DIMERIZATION OF THYMINE IN FROZEN SOLUTIONS*

Abstract— It is known that thymine forms dimers when aqueous solutions are irradiated with ultraviolet light while in the frozen state, but does not form dimers when solutions are irradiated in the liquid state. The eutectic point of aqueous thymine solutions was found to be. —0.02°C. Since the irradiation of frozen solutions is always carried out at lower temperatures, the dimerization must be occurring in the solid state. Activation energies and quantum yields for dimer formation were determined by irradiating 1–mm layers of thymine solution at —5°C to — 707deg;C for various lengths of time. As expected, the activation energy was zero. After measuring the amount of radiation scattered by samples of ice, the extreme values for the quantum yield were found to be 0.73 and 4.08. The lower limit assumed that all the scattered light was absorbed by thymine; the upper limit assumed that none was absorbed. Since the theoretical maximum quantum yield is 2, the best estimate of the quantum yield is considered to be between 1 and 2.     

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.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

2+2] Photocycloaddition reaction dynamics of triplet pyrimidines

Taking the 266 nm excited pyrimidine (uracil or thymine) with cyclopentene as model reaction systems, we have examined the photoproduct formation dynamics from the [2 + 2] photocycloaddition reactions of triplet pyrimidines in solution and provided mechanistic insights into this important DNA photodamage reaction. By combining two compliment methods of nanosecond time-resolved transient IR and UV-vis laser flash-photolysis spectroscopy, the photoproduct formation dynamics as well as the triplet quenching kinetics are measured. Characteristic IR absorption bands due to photoproduct formation have been observed and product quantum yields are determined to be ～0.91% for uracil and ～0.41% for thymine. Compared to the measured large quenching rate constants of triplet uracil (1.5 × 10(9) M(-1)s(-1)) or thymine (0.6 × 10(9) M(-1)s(-1)) by cyclopentene, the inefficiency in formation of photoproducts indicates competitive physical quenching processes may exist on the route leading to photoproducts, resulting in very small product yields eventually. Such an energy wasting process is found to be resulted from T(1)/S(0) surface crossings by the hybrid density functional calculations, which compliments the experiments and reveals the reaction mechanism.     

IMMUNOLOGICALLY ACTIVE LESIONS INDUCED ON DOUBLE-STRANDED DNA WITH ULTRAVIOLET

Abstract— Some immunochemical properties of double-stranded DNA irradiated with UV were studied, using a radioimmunoassay with irradiated [³H]-DNA and experimentally produced antibodies to DNA. Reactivity of antibodies revealed that irradiated DNA contained an immunologically active structure other than the irradiated DNA specific structure, resembling that of thermally denatured DNA. inhibition assay demonstrated that while DNA-antibody binding was effectively inhibited by mixed purine and pyrimidine oligonucleotides, thymine dimer containing pyrimidine oligonucleotides derived from the irradiated DNA showed no appreciable inhibition. The antigenic structure specific for irradiated DNA was found to be thermally labile in low salt medium. Cupric and ferrous ions and cysteine added to the DNA solution inhibited antigenicity formation during irradiation, but these substances exhibited no effect on dimer formation in irradiated frozen thymine solution. Calcium ions and histidine were inert for the former reaction but inhibited the latter effectively. This suggests that different mechanisms are involved in the 2 processes. The immunologically active UV-induced lesions appeared to depend mainly on a conformational structure change of the DNA strands rather than on a single modified base moiety.     

17O and 14N spectroscopy of 17O-labeled nucleic acid bases

Oxygen-17 and nitrogen-14 nuclear magnetic resonance techniques have been utilized in studying liquid-state hydrogen bonding interactions between nucleic acid bases and between the bases and solvent. The bases uracil and thymine have been labeled with ¹⁷O at positions 2 and 4, cytosine at position 2 and thymine riboside at position 4. The ¹⁷O chemical shift was found to be a sensitive structural probe and to provide information concerning the resonance forms that contribute to the total wave function. The chemical shift of the oxygen of thymine and uracil at position 2 was detected at a higher field than that of oxygen at position 4, presumably owing to an increased contribution of single bonded-oxygen valence bond structures at position 2. The ¹⁷O and ¹⁴N chemical shifts of cytosine were found to be strongly pH dependent, and the results have been interpreted in terms of predominant cytosine resonance structures contributing to the aqueous cation and anion. The ¹⁷O line width of cytosine in aqueous solution indicates the presence of hydrogen bonded dimers between neutral and cationic forms.     

PHOTOCHEMICAL REACTIONS OF AROMATIC KETONES WITH NUCLEIC ACIDS AND THEIR COMPONENTS I—. PURINE AND PYRIMIDINE BASES AND NUCLEOSIDES*.

Abstract— The photochemical reactions of benzophenone and acetophenone with purine and pyrimidine derivatives in aqueous solutions have been investigated by flash photolysis and steady-state experiments. Upon excitation of these two ketones in aqueous solutions, two transient species are observed: molecules in their triplet state and ketyl radicals. The triplet state lifetimes are 65 μsec for benzophenone and 125 μsec for acetophenone. The ketyl radicals disappear by a second order reaction, controlled by diffusion. In the presence of pyrimidine derivatives, the triplet state is quenched and the ketyl radical concentration is decreased without any change in its kinetics of disappearance. Ketone molecules in their triplet state react with purine derivatives leading to an increase in the yield of ketyl radicals due to H-atom abstraction from the purines. Steady-state experiments show that benzophenone and acetophenone irradiated in aqueous solution at wavelengths longer than 290 nm undergo photochemical reactions. The rate of these photochemical reactions is increased in the presence of pyrimidine derivatives and even more in the presence of purine derivatives. Following energy transfer from the triplet state of benzophenone to diketopyrimidines, cyclobutane dimers are formed. The energy transfer rate decreases in the order orotic acid > thymine > uracil. Benzophenone molecules in their triplet state can also react chemically with pyrimidine derivatives to give addition photoproducts. All these results are discussed with respect to photosensitized reactions in nucleic acids involving ketones as sensitizers.     

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.     

P-AMINOBENZOIC ACID CAN SENSITIZE THE FORMATION OF PYRIMIDINE DIMERS IN DNA: DIRECT CHEMICAL EVIDENCE

Thymine-containing photoproducts with chromatographic properties similar to those of cyclobutyl pyrimidine dimers can be formed in [³H]-thymine-labeled DNA in solution by 313 nm ultraviolet radiation in the presence of para-aminobenzoic acid (PABA), a compound used in sunscreen preparations. In the absence of PABA, similar fluences of 313 nm radiation do not produce significant numbers of these photoproducts. The thymine-containing photoproducts can be reversed by 254 nm radiation so that the tritium label migrates with the mobility of thymine monomer, a behavior characteristic of thymine-containing cyclobutyl pyrimidine dimers. This result supports previous, but less direct, data from other laboratories indicating that PABA can sensitize dimer formation in the DNA of bacterial and mammalian cells.     

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.