This survey focuses on recent developments in the far ultraviolet photochemistry of nucleic acids and related model compounds. The photoproducts discussed are the cyclobutidipyrimidines, the pyrimidine-pyrimidone adducts, the purine-pyrimidine adducts and the addition products of amino acids to pyrimidine bases. The specific aspects of the high-intensity laser photochemistry of nucleic acid components are also briefly reviewed.

Amino Acids↗

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Chemical structure of psoralen-nucleic acid photoadducts.

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5-Methoxypsoralen↗

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Use of morpholinonucleosides to conjugate oxidized DNA bases to proteins.

The universal method of Erlanger and Beiser was unsuccessful in conjugating 5-hydroxycytidine to proteins because of the instability of the base under the conditions used. A new strategy was developed to conjugate fragile modified nucleosides to proteins. This involves the use of morpholino derivatives of modified nucleosides. The validity of this method was demonstrated by the production of polyclonal antibodies specific for the DNA modification, 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Antibodies↗

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32P-postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide.

A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H2O2-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2'-deoxyadenosine N-1-oxide 3'-monophosphate and 2'-deoxyadenosine N-1-oxide 5'-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate of phosphorylation and to prevent self-radiolysis processes. [32P]-2'-deoxyadenosine N-1-oxide 5'-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close to 1 modified adenine N-1-oxide/10(6) bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H2O2.

Adenine↗

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Characterization of the cis-syn and cis-anti diastereoisomers of 5-methoxypsoralen pyrone-side monocycloadducts to thymidine.

The photoreaction of 5-methoxypsoralen (5-MOP) with thymidine as a DNA model compound was investigated under dry-state conditions. In this respect, a thin film of thymidine and 5-MOP in a ratio 10:1 was exposed to 350-nm UV light. Four [2 + 2] photocycloadducts were isolated in a 0.5-2.2% yield with respect to 5-MOP by HPLC and characterized as two pairs of cis-syn and cis-anti diastereoisomers, respectively, on the basis of extensive spectroscopic measurements, including UV, fast atom bombardment mass spectrometry, 1H and 13C NMR, and CD. Information concerning the absolute configuration of the four photocycloadducts was inferred from detailed nuclear Overhauser enhancement experiments. This is indicative of a 3R,4S,5R,6S and a 3S,4R,5S,6R configuration for the cis-anti cycloadducts and a 3S,4R,5R,6S) and a 3R,4S,5S,6R configuration for the cis-syn cycloadducts. In addition, conformational features of the four photocycloadducts were obtained from consideration of various 1H NMR measurements including NOE data.

5-Methoxypsoralen↗

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Reaction of singlet oxygen with 2'-deoxyguanosine and DNA. Isolation and characterization of the main oxidation products.

The reaction of singlet molecular oxygen with 2'-deoxyguanosine and DNA was studied. Emphasis was placed on the identification and characterization of the main methylene blue mediated type II (singlet oxygen) oxidation products of 2'-deoxyguanosine and its corresponding 3',5'-di-O-acetylated derivative. Two major oxidation products of 2'-deoxyguanosine were isolated and characterized by mass spectrometry analysis and extensive 1H and 13C NMR measurements as the two 4R* and 4S* diastereomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine. The addition of 1O2 was also found to occur to the base moiety of the corresponding 3',5'-di-O-acetylated derivative. Methylene blue mediated photosensitization of 2'-deoxyguanosine led also to the production of 7,8-dihydro-8-oxo-2'-deoxyguanosine, but in a relatively lower yield with respect to the two above diastereomers. The participation of singlet oxygen in the mechanism of formation of these oxidation products was confirmed. A reasonable mechanism involving the transient formation of an unstable endoperoxide produced through a Diels-Alder 1,4-cycloaddition of singlet oxygen to the purine ring is suggested. Quantitative analysis allowed us to demonstrate that the two diastereomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine are the main singlet oxygen oxidation products of the guanine moiety within nucleosides, whereas 7,8-dihydro-8-oxoguanine was found to be the major 1O2 oxidation product of guanine in double-stranded DNA.

Circular Dichroism↗

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Type I benzophenone-mediated nucleophilic reaction of 5'-amino-2',5'-dideoxyguanosine. A model system for the investigation of photosensitized formation of DNA-protein cross-links.

5'-Amino-2',5'-dideoxyguanosine has been synthesized in order to investigate the intramolecular reactivity of an amino group toward the guanine radical produced by type I photosensitization mechanism. Benzophenone-mediated photosensitization of 5'-amino-2',5'-dideoxyguanosine in aerated aqueous solution results in the formation of a predominant cyclic nucleoside together with an unstable nucleoside precursor. The two modified nucleosides have been isolated by reverse phase high performance liquid chromatography and characterized by spectroscopic measurements including 13C and 1H NMR, fast atom bombardment mass spectroscopy, and UV absorption. The stable photoproduct has been identified as 9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-7-ol, 3-amino- (1R-exo), whereas its precursor has been assigned as acetic acid, [(7-hydroxy-9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-3-yl)amino]oxo- (1R-exo). A reaction mechanism, involving nucleophilic addition of the sugar amino group to guanine radical intermediates, is proposed to explain the formation of the two photoproducts.

Benzophenones↗

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Gamma irradiation of 2'-deoxyadenosine in oxygen-free aqueous solutions: identification and conformational features of formamidopyrimidine nucleoside derivatives.

The two major radiation-induced decomposition products of 2'-deoxyadenosine in oxygen-free aqueous solution have been isolated by reverse-phase HPLC. The 1H and 13C NMR features of the two modified nucleosides obtained in DMSO-d6 are indicative of a similar formamidopyrimidine structure for the base residue (the ring-opened form of a C-8 hydroxylated purine). Interestingly, the sugar moiety exhibits a pyranose configuration, the two nucleosides being a pair of alpha and beta anomers. One-bond and long-range 1H-13C 2D NMR experiments have allowed the complete assignment of the carbon atoms. Confirmation of the base structure was obtained by 1H-15N scalar-correlated 2D NMR experiments. Attempts were made to characterize the expected furanose form of the initially generated formamidopyrimidine derivative. In this respect, isomerization reaction of the sugar moiety of the latter compound takes place rapidly after gamma-irradiation as inferred from 1H NMR analysis. The conformational study of the sugar moiety of the two pyranose anomers was inferred from detailed 600.13 MHz 1H NMR analysis in D2O. The alpha anomer exhibits a predominant 1C4 conformation whereas the beta anomer adopts preferentially a 4C1 conformation. In addition, the dynamic study of the restricted rotation of the formamido bond has revealed a 1/5 ratio in favor of the s-cis rotamer for both nucleosides. The energy barrier at coalescence was determined to be delta G# = 75.5 kJ.mol-1 (Tc = 370 K).

Carbohydrate Conformation↗

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An adduct between peroxynitrite and 2'-deoxyguanosine: 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2'-deoxyguanosine.

The reactivity of peroxynitrite (OONO-) with DNA is of interest because it is is released during chronic inflammation, a major contributor to cancer. Peroxynitrite was found to undergo homolytic addition to the C4-C5 double bond of 2'-deoxyguanosine with formation of 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2'-deoxyguanosine (nox-dG). This adduct may be a useful marker in the study of the chemical processes associated with the mutagenicity of peroxynitrite.

Chromatography, High Pressure Liquid↗

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Conformational and electronic properties of the two cis (5S,6R) and (5R,6S) diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine: X-ray and theoretical studies.

The structure of (+)-cis-(5S,6R)-5,6-dihydroxy-5,6-dihydrothymidine was obtained using X-ray crystallography [space group P2(1) with a = 10.130(3) angstroms, b = 6.434(9) angstroms, c = 11.02(5) angstroms, and beta = 112.646(2) angstroms]. The comparison of the two cis diastereoisomers of thymidine glycol (I, II) showed several structural and conformational differences. The solid state structures appear to be in agreement with the results of 1H NMR studies which were carried out in aqueous solution. Conformational and electronic properties of the ground state of the molecules I and II were obtained using ab initio LSD-DFT theory. Only slight differences between the crystal structure and the optimized geometry are observed for each of the two oxidized nucleosides. On the other hand, molecules I and II exhibit significant differences in their electronic properties. In particular, the dipole moment of (5S,6R)-thymidine glycol (I) is twice smaller than that of the (5R,6S) diastereoisomer (II). It is noteworthy that these differences in the electronic properties between the two compounds may be related to changes in the rotameric population around the C4'-C5' bond. The repartition of the electrostatic potential is different in the two compounds. These observations lead to a better understanding of the structural changes when the above lesions are included within a DNA molecule.

Crystallography, X-Ray↗