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The presence of a N-(2-deoxy-D-erythro pentofuranosyl)urea (henceforth referred to as deoxyribosylurea) residue, ring fragmentation product of a thymine, in a frameshift situation in the sequence 5'd(AGGACCACG).d(CGTGGurTCCT) has been studied by 1H and 31P nuclear magnetic resonance and molecular dynamics. At equilibrium, two species are found in slow exchange. We observe that the deoxyribosylurea residue can be either intra- or extrahelical within structures which otherwise do not deviate strongly from that of a B-DNA as observed by NMR. Our study suggests that this is determined by the nature and number of hydrogen bonds which this residue can form as a function of two possible isomers. There are two possible structures for the urea side chain, either cis or trans for the urido bond which significantly changes the hydrogen bonding geometry of the residue. In the intrahelical species, the cis isomer can form two good hydrogen bonds with the bases on the opposite strand in the intrahelical species, A4 and C5, which is not the case for the trans isomer. This results in a kink in the helical axis. For the major extrahelical species, the situation is reversed. The trans isomer is able to form two good hydrogen bonds, with G13 on the same strand and A7 on the opposite strand. For the extrahelical species, the cis isomer can form only one hydrogen bond. In this major structure the NMR data show that the bases which are on either side of the deoxyribosylurea residue in the sequence, G14 and T16, are stacked over each other in a way similar to a normal B-DNA structure. This requires the formation of a loop for the backbone between these two residues. This loop can belong to one of two families, right- or left-handed. In a previous study of an abasic frameshift [Cuniasse et al. (1989) Biochemistry 28, 2018-2026], a left-handed loop was observed, whereas in this study a right-handed loop is found for the first time in solution. The deoxyribosylurea residue lies in the minor groove and can form both an intra- and an interstrand hydrogen bond.

The synthetic deoxyoctanucleotide d(GTACGTAC) crystallizes as an A-type DNA double helix. The structure has been refined to an R factor of 17% at 2.4 A resolution with 56 solvent molecules located. The tetragonal unit cell, space group P43212 has dimensions a = 42.50 (7) and c = 24.79 (5) A. The asymmetric unit consists of a single strand of four base pairs.

1. The use of radioactive and biotinylated oligonucleotide probes has been optimized to detect and analyze byin situ hybridization, neurons expressing neuropeptide genes (vasopressin, oxytocin, somatostatin). 2. In situ hybridization was performed on cryostat-cut sections obtained from tissues perfused with 1% formaldehyde. Radioactive probes were labeled by tailing with35S-dATP and revealed with autoradiography. Biotinylated probes were obtained either by the incorporation of 11-biotin dUTP or by the addition of biotinylated nucleotides to the oligonucleotide during its synthesis. Biotin was revealed with streptavidin alkaline phosphatase and the appropriate substrate. 3. In the adult rat brain, radioactive and biotinylated probes revealed peptidergic neurons. The biotinylated probes provided an optimal cellular and subcellular resolution with a sensitivity similar to that observed with radioactive probes. Staining was selectively restricted to the cytoplasm and to the proximal part of processes. 4. Biotinylated vasopressin probes with 10 biotins added demonstrated magnocellular neurons and parvocellular neurons in the suprachiasmatic nucleus and the bed nucleus stria terminalis. 5. Vasopressin gene expression was studied during ontogeny in the rat fetus and neonate. Vasopressin mRNA was first detectable at gestational day 16 in the supraoptic nucleus in neurons of neuroblastic appearance. An aspect similar to the one present in adult was found at gestational day 19 in magnocellular neurons and at day 3 postnatal in parvocellular neurons. 6. The results confirm that radioactive oligonucleotide probes are efficient tools to investigate neuropeptide gene expression byin situ hybridization and demonstrate that biotinylated oligonucleotides are very efficient and provide a much higher resolution than radioactive probes with a reasonable sensitivity.

The processing of a unique uracil in DNA has been studied in mammalian cells. A synthetic oligodeoxyribonucleotide carrying a potential Bgl II restriction site, where one base has been substituted with a uracil, was inserted in the early intron of SV40 genome. Various heteroduplexes were constructed in such a manner that the restitution of an active Bgl II restriction site corresponds in each case to the specific substitution of the uracil by one of the four bases normally present in the DNA. DNA cuts by this restriction enzyme in one or several constructed heteroduplexes immediately determine the type of base pair substitution produced at the site of the U residue. When the uracil is inserted opposite a purine it is fully repaired; when facing a guanine it is replaced by a cytosine and opposite an adenine it is replaced by a thymine. These results indicate the error-free repair of uracil when it appears in the cell with the usual mechanisms such as cytosine deamination or incorporation of dUTP in place of dTTP during replication. When the uracil is inserted opposite a pyrimidine no error free repair at all is detected for U:C or U:T mismatches. It appears, moreover, that in approximately 18% of the cases U:T mismatch leads to a C:G base pairing. In the majority of the U:pyrimidine mismatches, mutations occur in the vicinity of the uracil, including base substitutions and frameshifts by addition of one or several bases.

Deoxyribosylurea is an alkali sensitive DNA defect induced by the action of ionizing radiation. Oligonucleotides bearing this residue were synthesized using 5′-O-(4-methoxytrilyl)-2′-deoxyribosylurea-3′-O-(2-cyanoethyl-N,N'-diisopropyl) phosphoramidite and PAC protected deoxynucleoside phosphoramidites.

New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.

The present study has been undertaken to investigate the efficiency of biotinylated synthetic oligonucleotide probes in detection by in situ hybridization of the mRNAs coding for calcitonin (CT) or calcitonin gene-related peptide (CGRP) in human medullary thyroid carcinomas (MTCs). Tissue sections fixed with formaldehyde were hybridized with 45-base long oligonucleotides, specific for CT or CGRP mRNA. Recombinant DNA probe or synthetic oligonucleotides radioactively labelled with 32P or 35S were used as controls to detect by autoradiography the corresponding mRNAs in the tumour cells. Oligonucleotide probes labelled by fixation of one biotin molecule at their 5'-end, or by incorporation of a tail of biotin-11-dUTP at their 3'-end, were used and were revealed by incubation with streptavidin-alkaline phosphatase associated with the corresponding substrate. Each biotinylated probe stained exclusively the cytoplasm of the tumour cells, the CT probe giving a much higher level of staining than the CGRP probe. The same cells were found to contain CT and CGRP mRNAs. Controls performed with either radioactive or biotinylated probes confirmed the specificity of the staining. These results demonstrate that biotinylated synthetic oligonucleotides can be used as efficient tools to investigate gene expression in tissue sections, thus avoiding the various inconveniences connected with the use of radioactive probes, especially bio-hazards, the use of autoradiography, the limited histological resolution, and the delay in obtaining results.

N-formamido-(DNA) is one of the main lesions formed on the thymidylic fragments of DNA when DNA, in vitro or in vivo conditions, is exposed to ionising radiations. The N-formamido group partly replaces the thymine base in DNA sequence. This article proposes an indirect dosing technique for this damage. The method is based on the release of 14CO2 from the C-2 of the thymine ring. When the pyrimidine ring of the thymidine moiety is broken, N-formamido deoxyribose derivative, carbon dioxide and pyruvamide are formed all in the same reaction. During this investigation the phenomena of protection due to the constituent parts of the cell and to the secondary and tertiary structures of DNA are also evidenced.

Resume Les (+) et (−) l'hydroxy-6-dihydro-5,6-thymidines cis 4a, 5a sont prepares par reduction douce des formes (+) et (−) de la brorno-5-hydroxy-6-dihydro-5,6-thymidine trans 2a, 3a. Les “hydrates” trans 6a, 7a sont obtenus par chauffage de 4a ou 5a en solution aqueuse alcaline a 60°. L'isomerisation de 4a, 5a, 6a et 7a implique l'ouverture du cycle pyrimidique en 1,6, suivie d'une reaction de ceto-enolisation. La determination de la configuration des “hydrates” de thymidine repose sur l'obtention specifique des formes enantiomeres de l'hydroxy-6-dihydro-5,6-thymine par degradation radio-induite de 4a, 5a, 6a, 7a et de la dihydro-5,6-thymine 5S 8b.

The presence of a m6A-T base pair does not give rise to a major change in helix conformation from that of a normal B DNA, but does slow down dramatically the rate of helix formation.

To determine the radiation sensitivity of an adenine defect inside the DNA chain, an oligonucleotide bearing 7,8-dihydro-8-oxoadenine was chemically synthesized by the phosphoramidite method. This damaged oligonucleotide 32P-labelled at one end was gamma irradiated in an aerated aqueous solution, and heated with piperidine. The fragments obtained were separated by polyacrylamide sequencing gel electrophoresis, according to their chain lengths. Mean radiosensitivity coefficients for the four natural nucleotides and the relative radiation sensitivity of 7,8-dihydro-8-oxoadenine leading to chain rupture in alkaline medium, could be obtained from the band intensities by computer calculation. An unexpectedly high value was obtained for the damaged adenine residue. Determinations were performed for different doses of irradiation.

The radiolysis products resulting from the cleavage of the N-glycosidic linkage after gamma irradiation of cytosine 14C-2 labelled DNA of Escherichia coli were mainly cytosine and trans 5, 6-dihydroxy-5, 6 dihydro uracil. Relative percentages of these products were determined against doses and mechanism of release suggested.

Deoxyoligonucleotides 32P-labelled at one end have been used to probe the DNA chain breakage induced by external gamma-irradiation and alkali treatment. The fragments were separated according to their chain lengths by polyacrylamide gel electrophoresis and their radioactivity counted. Quantitative analysis of the distribution pattern of the intensity fragments was performed by computer. In this article the principles of the calculation program are given. The basic units corresponding to adenine, thymine, cytosine and guanine exhibited different radiosensitivities. It was possible to estimate the mean chain rupture per nucleotide and per Gray for G, T, C and A. These coefficients were derived from experimental values by iteration. Thus, it is possible to simulate very accurately the oligonucleotide fragment intensities. The relative error was usually less than 10 per cent for most of the nucleotide units. In this way small differences in the band intensities may be demonstrated. They can be explained by variations of the local conformation of the biopolymer.

The final radiation products obtained by gamma-irradiation of frozen aqueous solutions of thymidine have been identified as 5,6-dihydro-5,6-dihydroxythy-midine, 5,6-dihydrothymidine, thymidine dimers, 1-(2-deoxy-beta-D-threo-pento-furanosyl)-thymine, 1-(2-deoxy-alpha-L-threo-pentofuranosyl)-thymine, thymine and 5,6-dihydrothymine. The nature of the radiation products could be explained on the basis of the radical structures reported earlier.

Radicals determined by e.s.r. spectrometry of irradiated thymine or thymidine and radiolytic products generated under tha ction of gamma rays in aerated aqueous solutions have been compared. This comparison lies mainly in the fact that a radical R gives rapidly the corresponding peroxide ROOH. The authors have isolated and characterized twenty peroxides, i.e., the four isomers cis (-), cis (+), trans (-), trans(+) of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine; the four isomers cis (-), cis (+), trans (-), trans (+) of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine; 5-hydroperoxy-2-deoxyuridin;cis and trans 6-hydroperoxy-5-hydroxy-5,6-dihydrothymine; cis and trans 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine; 5-hydroperoxymethyl-uracil; 5-hydroperoxy-5,6-dihydrothymine;cis and trans 6-hydroperoxy-5,6-dihydrothymine; 5-hydroperoxy-5-methyl barbituric acid; 5-hydroperoxy-5-methyl hydantoin; trans 5,6-dihydroperoxy-5,6-dihydrothymine. Most of thethymine and thymidine radicals hypothesized or described in the literature were correlated to these peroxides. However, the presence of certain peroxides could not be explained by recognized radicals. Taking advantage of this fact, the existence of new thymine or thymidine radicals so far unknown can be predicted.

Recent data on the radiation chemistry of 2'-deoxyribonucleosides are discussed. Various mechanisms such as dismutation, charge transfer, and dimerization are suggested to explain the formation of 5, 6- dihydrothymidine, its various 5- and/or 6-hydroxylated derivatives, and noncyclobutane-type dimers. Additional information on these complex processes has been obtained using radical scavengers and radio sensitizing agents. (The latter act mainly by electron transfer and binding reactions). These studies have also been extended to purine nucleosides. In particular, specific reactions have been studied for the radiation-induced sugar radicals of 2'-deoxyguanosine.

Resume La preparation d'un pentadecanucleoside tetradecaphosphate contenant la base modifiee dihydro-5,6 thymine (DHT) est rapportee. Cette synthese est effectuee a partir d'un melange de diastereoisomeres 5R et 5S du nucleoside, obtenu par hydrogenation catalytique de la thymidine. Le synthon phosphoryle, destine a l'assemblage, a ete caracterise par RMN du proton et par spectrometrie de masse (FAB). Il a ete incorpore dans un fragment de DNA (oligo dT) par la methode phosphotriester en phase solide. Apres deprotection, la caracterisation et la localisation du defaut ont ete effectuees selon plusieurs approches. Par la methode de sequencage de Maxam et Gilbert, il n'a pas ete possible de differencier les residus thymines du pentadecamere. La DHT a ete mise en evidence par une reaction specifique de chauffage a 90°C dans l'acide formique. Enfin, la spectrometrie de masse en impact electronique dans des conditions pyrolytiques permet d'affirmer que la base modifiee est presente dans l'oligonucleotide deprotege.

Starting with 4-thio-2′-deoxyuridine, the simple synthesis of N 4 -mono- and dialkyl-2′-deoxycytidines is described. The N 4 -methyl derivative has been incorporated into the oligonucleotide chain d(CGm 4 CGCG).

The mechanism of the epimerization of 5,6-dihydroxy-5,6-dihydrothymine was studied using 18O labeled compounds (positions 5 or 6) prepared by specific syntheses. Results provided by mass spectrometry were in agreement with a mechanism involving a N1C6 ring chain tautomerism. Specific isomerization of (+) cis thymine glycol into its (-) trans form was observed.

Radiolysis products resulting from the deamination pathway were isolated and identified after irradiation of cytosine by gamma rays in neutral aqueous solutions. They were identical to those identified in uracil radiolysis i. e. 5,6-dihydroxy-5,6-dihydro-uracil (cis and trans forms), iso-dialuric acid, alloxan, 5-hydroxy-hydantoin, parabanic acid and oxaluric acid. The absence of 4,4′-di-iso-barbituric acid, alloxantin or iso-barbituric acid could be explained by the good stability of uracil glycols. The identification of buiret proof of potential degradation mechanisms different from these involving of the free radical attack on the 5,6 double bond. Gamma Irradiation of Cytosine in an Aerated Aqueous Solution, I

SummaryLe comportement de l'acide uridylique sous rayonnement γ en solution aqueuse a la concentration 10−4 M (pH∼6, 14C-4) a ete etudie. En utilisant des methodes chromatographiques sur couche mince de silice associees a des mesures spectroscopiques (spectrometrie de masse, I.R., R.M.N.) nous avons identifie huit substances dont le G de formation a ete calcule pour 16 krads. Ce sont les dihydroxy-5,6-dihydro-5,6 uracile cis (G = 003) et trans (G = 005), l'acide isodialurique (G = 001), l'hydroxy-5 hydantoine (G = 001), l'alloxane (G traces), l'acide parabanique (G traces) et la N-formyl N′-glyoxyl uree (ou N-formyl hydroxy-5 hydantoine) (G traces), l'uracile (G = 028). Il apparait que la rupture de la liaison N-glycosidique est un phenomene important; la presence de ribonolactone phosphate, decelee indirectement, permet d'expliquer un mode de coupure de cette liaison apres attaque au niveau du carbone 1′.La base est la partie la plus radiosensible vis a vis de l'action des radiacux OH.; cependant, le rib...

Nous presentons und serie de spectres de masse originaux, de derives de l'uracile et de la thymine, ainsi que les schemas de fragmentation correspondants. L'etude d'une premiere serie de molecules, a caractere aromatique, dont une marquee au 14C, a permis de confirmeer le mecanisme de base de la fragmentation comme etant un ‘retro Diels Alder’. Une deuxieme serie, Saturee en positoin 5,6 du noyau, montre le caractere totalement different de ces molecules, plus fragiles, plus sensibles a la nature des substituants et donnant un ion protone [MH]+, responsable d'une partie des fragments observes. Original mass spectra of uracil and thymine derivatives are presented with the corresponding fragmentation schemes. In the first series of spectra, the fragmentations of thymine derivatives, including 14C-2-thymine, dimers, and bromo thymines, confirm the basic retro Diels-Alder mechanism. The second series includes dihydro 5,6-derivatives of uracil (hydroxy and bromo substituents). The behaviour of these molecules is quite different; they are more sensitive to the substituents and a part of the fragmentation is often explained by protonated molecular ions.

Peroxides produced from thymine by γ irradiation in aerated solution 1. 1. Chemical modifications of DNA upon γ irradiation play an important role in radiobiology. Peroxides produced from thymine by γ irradiation in aerated solution have been studied. Thymine hydroperoxides were the primary products and their chemical structures which were mostly unknown, have been determined. 2. 2. The following hydroperoxides were identified: ◦ trans -5-Hydroperoxy-6-hydroxy-5,6-dihydrothymine ◦ cis -5-Hydroperoxy-6-hydroxy-5,6-dihydrothymine ◦ cis -6-Hydroperoxy-5-hydroxy-5,6-dihydrothymine ◦ 5-Hydroperoxymethyl uracil ◦ 5-Hydroperoxy-5,6-dihydrothymine ◦ cis -6-Hydroperoxy-5,6-dihydrothymine ◦ trans -6-Hydroperoxy-5,6-dihydrothymine ◦ trans -5,6-Dihydroperoxy-5,6-dihydrothymine ◦ 5-Hydroperoxy-5-methylhydantoin ◦ 5-Hydroperoxy-5-methylbarbituric acid 3. 3. These compounds were separated by thin-layer chromatography involving new chromatographic solvents. By decomposition they were converted into alcohol besides other degradation products. 4. 4. These 14 C-labelled peroxides were synthesized by different methods which are described. A new way to obtain thymine peroxides based on the reaction of H 2 O 2 with halogenated derivatives of thymine, in the presence of Ag 2 O is given. 5. 5. Spectral analysis (ultraviolet, infrared, NMR, mass spectrometry) gave information on the configuration and the conformation of the products. Chemical couplings and J constants provided by NMR spectrometry were tabulated. 6. 6. Quantitative analysis of peroxide formation and decomposition products was performed by 14 C scintillation counting. 7. 7. The peroxides obtained at pH7 result from the action of oxygen on five different radicals: ◦ 5-Hydroxy-5,6-dihydro-6-thymyl ◦ 6-Hydroxy-5,6-dihydro-5-thymyl ◦ (5-Uracyl)methyl ◦ 5,6-Dihydro-5-thymyl ◦ 5,6-Dihydro-6-thymyl The isolation of peroxides is proof of the existence of these radicals which could be detected under different experimental conditions by EPR spectrometry. The addition of O 2 or O − 2 to the 6-hydroxy-5,6-dihydro-5-thymyl radical is preferentially performed in the trans configuration.

Resume Les deux isomeres de l'hydroxy-6 dihydro-5,6 thymine, forme cis et forme trans, ont ete obtenus par irradiation gamma de la thymine en solution aqueuse desaeree (2 . 10 −3 M) avec du 60 Co. Leurs proprietes chimiques et chromatographiques, leurs spectres infra-rouge et de resonance magnetique nucleaire, leurs syntheses chimiques sont decrites. De plus, la formation de ces produits sous rayonnement a ete etudiee en comparaison avec celles des autres substances de radiolyse de 9000 a 10 6 rads. Les substances hydrogenees comme la dihydrothymine apparaissent en quantites importantes. Les “hydrates” de thymine sont susceptibles de prendre part dans les phenomenes de thermorestauration des cellules vivantes irradiees.

L'etude du spectre de masse des anomeres pyranniques et furanniques de la thymidine, de leurs acetates et des bromohyrines correspondantes apporte des informations, interessantes pour l'analyse de al configuration anomerique de ce type de composes, decisives pour la dimension de l'heterocycle oxygene. La stabilite des ions molecularies va de pair avec la stabilite chimique de ces molecules: les ions moleculaires des formes β sont plus ceux des formes α. L'instabilite des ions moleculaires des bromohydrines est telle qu'ils ne sont detectables que pour la forme β furannique. L'elimination du radical CH2OH ou CH2OAc est caracteristique de la forme furannique. L'elimination de CH2O dans la forme β furannique est due a la proximite sterique de la base et de l'hydroxyle en 5′ du sucre. Les ions de rearrangement [BH CHCH2]+ et derives proviennent d'und rupture du sucre favorisee dans la forme furannique. Apres cassure de la liaison glycosidique, les ions [B + H]+˙ et [B + 2H]+ formes sur la base, et [S]+ sont sensibles a la stereochimie de la molecule. L'´elimination d'une molecule d'eau (ou d'acide ac´etique) est mormalement plus facile dans un cycle a 6 chainons (pyrannique). L'elimination simultance de 2 molecules d'eau ou d'acide acetique, preponderante dans la forme furannique, nous a conduit a conduit a admettre und rupture du cycle pour former un ion a sttructure aliphatique conjuguee; la forme pyrannique donne un ion cyclique aromatique, en 2 etapes. The mass spectra of furanic and pyranic anomers of thymidine as well as those of acetates and bromohydrines of these four molecules are studied and compared. The stability of molecular ions is correlated with the chemical stability of these copmpounds. Molecular ions of β forms are more stable than those of α forms. The instability of the bromohydrines molecular ions is such that they are only observed for β furanic anomers. The eliminmation of the ·CH2OH or ·CH2OAc radical is noterd only for the furanic compound. The cleavage of CH2O in the β furanic substances is correleated with the steric vicinity of the base and the sugar 5′ hydroxide. The ions[BH ChCH2]+ and those resulting from this rearrangement and cleavage of sugar moiety, are enhanced in the case of furanic forms. After rupture of the N-glycosidic bon, the [B + H]+˙ and [B + 2H]+ ions resulting from the base and [S]+ are related to the molecular stereochemistry. The eliminationof one water molecular(or acetic acid) is easier in a sixs membered ring(pyranic). The concerted elimination of two water molecules (or acetic acid), more important in the furanic form, enables us to establish a ring cleavage mechanism which gives an ion with a conjugated aliphaticf structure; the pyranic form gives an aromatic cyclic ion in two steps.

On expose un ensemble de resultats experimentaux concernant la polymerisation radiochimique de ľethylene sous rayonnement gamma, effectuee en deux phases successives: initiation sous flux eleve (jusqu'a 105 rads/h) a des temperatures comprises entre -40°C et +22°C. propagation sous faible flux (225 rads/h) a une temperature de 12°C. Les caracteristiques des polymes obtenus (masses rnoleculaires, densites, point de fusion, indice de branchement) sont donnees. On etudie ľinfluence de la temperature pendant la phase ďinitiation pour: la cinetique de propagation, les masses moleculaires des produits obtenus, les rendements radiochimiques. Elle est beaucoup plus sensible sur le rendement radiochimique de propagation que sur le rendement radiochimique ďinitiation. MECHANISM OF GAMMA RADIATION POLYMERIZATION OF ETHYLENE The following two-step reaction was used to obtain further information on the mechanism of gamma radiation polymerization of ethylene: First period: Initiation reaction with 105 rads/h a...

In gamma-irradiation of cytosine in aerated aqueous solution, two new radiolysis products were isolated and identified: 1-carbamoyl-5-hydroxyhydantoin and N-formylbiuret. This formation involves ring cleavage between C4 and C5 and belongs to the nondeamination pathway.

Resume La cyclo-5′,8 desoxy-2′ adenosine 2 a ete identifiee comme l'un des principaux produits de la radiolyse gamma de la desoxy-2′ adenosine 1 (G = 0.03 en presence d'oxygene, G = 0.05 en absence d'oxygene). Cette substance est susceptible de jouer un role dans les phenomenes de mutagenese radio-induites. Les etudes de 1 H-RMN a 250 MHz ont mis en evidence la geometrie remarquable de ce compose puisque trois constantes de couplage de protons vicinaux du fragment sucre ne sont pas detectees. L'etude du modele montre en effet que les protons correspondants forment un angle voisin de 90°. La formation de 2 implique l'attaque de la double liaison C8-N7 par le radical en 5′ de 1 produit sous l'action des radicaux OH· issus de la radiolyse de l'eau.

(1) “Uridine hydrates” i.e (+)- and (−)6-hydroxy-5,6-dihydrouridine were formed under gamma irradiation in a deaerated aqueous solution of uridine. (2) The structures of two diastereoisomers were determined by spectroscopic measurements (infrared, ultraviolet and NMR) and verified by stereospecific synthesis; uridine hydrates were prepared by mild reduction of trans (+)- and (−)iodohydrins with acetic acid and zinc powder. (3) The carbon 6 epimerisation of uridine hydrates 6R ∗ or 6S ∗ was performed in tritiated water (pH 5.5, 30° C) and at the same time tritium incorporation on carbon 5 was noted. The mechanism of these reactions could be explained by the opening of the N 1 -C 6 bond of the pyrimidine ring, followed by ketoenolisation reaction of carbons 4 and 5. (4) The 250 MHz NMR analysis has allowed us to determine the nucleoside conformations. Nucleosides had mainly the S(C 2 ′ endo) conformation. A slight preference for gauche-gauche (gg) rotamer of the exocyclic hydroxymethyl group was noted and the aglycone was in the anti conformation.

Electrophoresis on polyacrylamide gels of the fragments resulting from gamma-irradiation of single-stranded oligodeoxyribonucleotides labelled at their 5'- or 3'-end proved to be a potent tool for the analysis of the radiation-induced chain breakage of DNA. Owing to the fact that the oligonucleotide may be ruptured at more than one site, the counting of the electrophoresis bands must be corrected and it is necessary to assess the influence of the cleavage position on the band intensities. A complicating factor is the inhomogeneity of the system due to the presence of the four bases A, T, C and G. To circumvent this problem, the homooligodeoxyribonucleotides (dA)15, (dC)15, (dT)15 were used as experimental probes. They were gamma-irradiated in solution, heated in alkali and the resulting fragments separated by gel electrophoresis. A computer simulation of the band intensities was compiled based on the general assumption that the chain breakage is homogeneous. The experimental results obtained from the homooligodeoxyribonucleotides labelled at either the 5' or the 3'-end are in excellent agreement with theoretical calculations. Abacus giving the gel band intensities (percentage) against the nucleotide positions and the remaining intensity of the original oligonucleotide have been obtained.

Radiosensitizers react with nucleic radicals by addition and by electron transfer reactions. We report the steady-state gamma radiolysis of 1 mM thymine in oxygen-free aqueous solutions containing different classes of radiosensitizing drugs: N-oxyl-free radicals (TAN and TMPN), quinones (menadione and naphtoquinone), nitroheterocyclic compounds (metronidazole and 5-nitro-2-furoic acid) and N-ethylmaleimide. Two classes of thymine degradation products were isolated by thin-layer chromatography and characterized by spectroscopic measurements. The main products, irrespective of radiosensitizers, resulting from oxidation reaction were identified as the cis and trans isomers of 5,6-dihydroxy-5,6-dihydrothymine, N-pyruvyl-N'-formylurea, 6-hydroxy-5,6-dihydrothymine and 5-hydroxy-5,6-dihydrothymine. In the experimental conditions used only N-oxyls and to a lesser extent NEM reacted with 5-hydroxy-5,6-dihydrothymin-6-yl radical, giving stable covalently-bonded addition products with a high yield. TAN showed a higher binding ability with respect to TMPN, which is in good agreement with the rate-constants previously reported for these bimolecular reactions.