E.s.r. of spin-trapped radicals in gamma-irradiated aqueous solutions of nucleic acids and their constituents.

The gamma-radiolysis of de-aerated neutral aqueous solutions of uracil, thymine, cytosine and of the corresponding nucleosides and nucleotides and of calf-thymus DNA was investigated. For uracil and thymine, the U.V. photolysis of aqueous solutions containing H2O2 was also studied. The short-lived radicals were spin-trapped by tert-nitrosobutane and identified by electron-spin-resonance spectroscopy. For all compounds two or more radicals were observed, and these could be distinguished by following the thermal decay of the spin adducts. Radicals formed by the addition of H or OH at the C(5) or C(6) positions of the pyrimidine derivatives were observed in all cases. Sodium formate was used as a scavenger for H and OH to identify the radicals formed by eaq-. Spin-trapped radicals in gamma-irradiated aqueous solutions of polynucleotides exhibited broad e.s.r. lines. For DNA gel, additional narrow lines due to scission products were also found.     

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.     

E.s.r. of free radicals in aqueous solutions of substituted pyrimidines.

Reactions of OH radicals with methyl and ethyl derivatives of uracil, cytosine and thymine in aqueous solutions have been investigated. Photolysis of H2O2 was used to generate OH radicals and the radicals on the base derivatives were spin-trapped using t-nitrosobutane and identified with the help of e.s.r. spectroscopy. Addition of OH radicals was found to take place predominantly to the C(5)--C(6) double bond of the bases. H-abstraction from the methyl group occurred in the N(1) methyl derivatives of uracil, cytosine and thymine. Radicals formed by H-abstraction from the methyl group were also detected for 3-methyluracil, thymine, 1-methylthymine and 1-ethylthymine. Introduction of a methyl or ethyl group at the N(1) position of uracil, cytosine and thymine causes an increase in the C(6) proton coupling and a decrease in the N(1) splitting for radicals formed by OH addition at the C(5) position.     

The energetics of rearrangement and water elimination reactions in the radiolysis of the DNA bases in aqueous solution (eaq- and *OH attack): DFT calculations

DFT calculations on the relative stability of various nucleobase radicals induced by e(aq)(-) and (*)OH have been carried out for assessing the energetics of rearrangements and water elimination reactions, taking the solvent effect of water into account. Uracil and thymine radical anions are protonated fast at O2 and O4, whereby the O2-protonated anions are higher in energy (50 kJ mol(-1), equivalent to a 9-unit lower pK(a)). The experimentally observed pK(a)=7 is thus that of the O4-protonated species. Thermodynamically favored protonation occurs slowly at C6 (driving force, thymine: 49 kJ mol(-1), uracil: 29 kJ mol(-1)). The cytosine radical anion is rapidly protonated by water at N3. Final protonation at C6 is disfavored here. The kinetically favored pyrimidine C5 (*)OH adducts rearrange into the thermodynamically favored C6 (*)OH adducts (driving force, thymine: 42 kJ mol(-1)). Very similar in energy is a water elimination that leads to the Ura-5-methyl radical. Purine (*)OH adducts at C4 and C5 (plus C2 in guanine) eliminate water in exothermic reactions, while water elimination from the C8 (*)OH adducts is endothermic. The latter open the ring en route to the FAPY products, an H transfer from the C8(*)OH to N9 being the most likely process.     

Photoinduced reactions of anthraquinone antitumor agents with peptides and nucleic acid bases: an electron spin resonance and spin trapping study

The photoexcitation (lambda = 313 +/- 10 nm) of adriamycin, daunomycin, and mitoxantrone in the presence of peptides or pyrimidine nucleic acid bases was investigated. In air-saturated and air-free solutions, peptides are decarboxylated by the photoexcited drug molecules. The decarboxylation reactions were shown to occur specifically at the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin-trapped using 2-methyl-2-nitrosopropane (MNP) and identified by electron spin resonance (ESR). In air-free solutions, nucleic acid bases are oxidized by the photoexcited drug molecules predominantly generating C(5)-carbon-centered radicals in the pyrimidine rings of uracil, cytosine, and thymine. However, spin adducts of MNP and thymine were also obtained at the N(1) or N(3) positions of the pyrimidine ring. In air-saturated adriamycin and daunomycin solutions, the spin adducts of MNP with uracil or thymine are similar to those obtained following hydroxyl radical reactions with these pyrimidines. This suggests that in the presence of oxygen, the photoexcited adriamycin and daunomycin transfer an electron to oxygen generating the superoxide anion radicals (O2-.), which are precursors of hydroxyl radicals. O2-. was also formed when O2-saturated DNA solutions were photoirradiated (lambda = 313 +/- 10 and 438 +/- 10 nm) in the presence of adriamycin and daunomycin, indicating that the photodegradation of DNA in the presence of these drugs caused by hydroxyl radicals is mediated by dissolved oxygen.     

The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU)

Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.     

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.     

Compounds, similar to acyclovir V. Synthesis and antiviral activity of carbethoxyalkoxymethyl derivatives of nucleic bases

Ethyl esters and amides of carboxymethoxy-, alpha- and beta-carboxyethoxy- and gamma-carboxypropoxymethyl derivatives of adenine, guanine, cytosine, uracil, thymine, 1,2,4-triazole-3- and -5-carboxamide were synthesized and their antiviral activity was studied.     

Hydrogen-bonding preferences in 2,6-diaminopurine: uracil (thymine) and 8-methyl adenine:uracil (thymine) complexes

We present molecular mechanical calculations on 2,6-diaminopurine (2,6-DAP):uracil (thymine) and 8-methyladenine (8-methyl A): uracil (thymine) hydrogen-bonded complexes of various geometries, namely, Watson-Crick (normal and reverse), Hoogsteen (normal and reverse), and purine N3 type. In contrast to earlier calculations [Ornstein, R. L. & Fresco, J. R. (1983) Proc. Natl. Acad. Sci. USA 80 , 5171–5175], the 2,6-DAP:uracil (thymine) complexes are predicted to be Watson-Crick and the 8-methyladenine:uracil (thymine) to be Hoogsteen. The results presented here are more consistent with the observed crystallographic preferences.     

Chlorination studies. IV. The reaction of aqueous hypochlorous acid with pyrimidine and purine bases

The labile intermediates and stable end products formed by the reaction of aqueous HOCl with thymine, uracil, 5-Br-uracil, N,N-dimethyluracil and 6-methyluracil have been identified. The purine ring system of guanine, adenine and xanthine was more resistant to attack by aqueous HOCl and reaction times of one week resulted in the formation of parabanic acid. Caffeine and theophylline under similar reaction conditions yielded N,N-dimethylparabanic acid.     

Interfacial behavior of uracil derivatives

The adsorption of a number of methylated uracil derivatives and of 5-fluorouracil has been studied by surface electrochemical methods at a mercury electrode. All derivatives exhibit an initial or dilute adsorption region where they are adsorbed flat on the electrode surface and are bound by pi-electron overlap with the electrode. Uracil, thymine, 1,5-dimethyl-uracil, 5,6-dimethyluracil, 1,5,6-trimethyluracil and 5-fluorouracil undergo a surface reorientation from the initial flat solution activities for each compound. An unsubstituted N(3)-H group is an absolute requirement for a uracil derivative to be capable of adopting the perpendicular surface stance. In the perpendicular orientation the uracil derivative appears to be bound to the electrode primarily via a N(3)-H--(-) electrode bond although a similar but weaker hydrogen bond can be formed via the N(1)-H group for certain compounds.     

Reaction of Bromotrichloromethane Derived Free Radicals with Uracil in a Model System. Structures of Products Formed

Free radicals generated by benzoyl peroxide-mediated catalytic decomposition of bromotrichloromethane (eg. trichloromethyl) were allowed to react under nitrogen or under air with uracil. Under nitrogen two reaction products were formed, one was identified as 5-chlorouracil and the other as a 5-bromouracil. Under air, besides the above two products other nine were also formed: 5,6-dihydrouracil; 5-hydroxyuracil; a chlorohydroxy adduct of uracil; a bromohydroxy derivative of uracil having the 5,6 bond in the saturated form; other bromohydroxy derivative of uracil having the double bond intact; 5,6-dihydroxyuracil; two dihalogenated hydroxylated uracil derivatives and one peak we were not able to descipher its structure. No single reaction product formed had carbon centered radicals (eg. trichloromethyl) added from CBrCl₃ and consequently would be missed in 'in vivo' covalent binding studies where ^l4C haloalkane (CBrCl₃ or carbon tetrachloride) were employed. If similar reaction products resulted during interaction of CBrCl₃ reactive metabolites with uracil in RNAs, significant deleterious effects in their function would be expected. That possibility, however, remains to be established.     

Effects of 5-azacytosine in DNA on enzymic uracil excision

PBS-2 phage DNA, which contains uracil in place of thymine, was used as substrate for both purified B. subtilis uracil-DNA glycosylase and a crude extract from M. luteus. Addition of [3H]5-azacytidine to the medium after phage infection resulted in substitution of 1.2% azacytosine for cytosine in DNA. Substrate DNA was also labeled with [14C]uracil. Neither enzyme preparation released tritiated bases from DNA. Analysis by S1 nuclease digestion show no increase in single-strandedness of the modified DNA. Enzymic release of uracil by the M. luteus extract was reduced by about 50% from the substituted substrate. By contrast, the rate of uracil excision by the purified enzyme was unaffected by the presence of DNA 5-azacytosine.     

Potential and structure controlled interfacial behavior of uracil derivatives

The adsorption and related interfacial behavior of uracil, various methylated uracil derivatives, uridine, uridine-5'-monophosphate and uridine-3'5'-cyclic monophosphate has been studied by surface electrochemical measurements at a mercury electrode. All uracil derivatives exhibit an initial "dilute" adsorption region where the virtually flat uracil residue is absorbed flat on the electrode surface. In the case of uracil and its methylated derivatives the area occupied by one molecule is about 60-70 A2. Uracil, thymine and 1,5-dimethyluracil exhibit a second adsorption region where they rearrange on the surface and adopt a perpendicular orientation and occupy about 40 A2 per molecule. In this perpendicular orientation the uracils are bound to the electrode through the N(3)-H or perhaps N(1)-H functions in a manner similar to their Watson-Crick bonding in nucleic acids. When in the perpendicular orientation the adsorbed molecules undergo extensive stacking (association) interactions, again similar to those observed between adjacent bases in nucleic acids. The ability of a uracil derivative to undergo a surface reorientation is critically dependent on electrode potential, bulk-solution concentration and molecular structure.     

Products distribution in the oxidation of thymine with hydroxyl radicals

In the oxidation of thymine with hydroxyl radical generated from L-ascorbic acid/copper(II) ion/O system, four reaction products, thymine glycol(TG), N-formyl-N'-pyruvylurea(FPU), 5-hydroxymethyluracil(HMU) and 5-hydroxy-5-methyl barbituric acid(HMBA) were obtained. A reaction scheme was proposed to explain the products distribution observed.     

A new and efficient strategy for the synthesis of shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine and -uridine

Two shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine (2) and -uridine (3) have been synthesized using D-arabinose, L-fucose, thymine, uracil, and stearoyl chloride as the starting materials. The synthetic procedures involve the facile preparation of 1-(3,5-di-O-benzyl-beta-D-ribofuranosyl)thymine (9) and -uracil (10) by coupling of 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) with silylated thymine and uracil, and then stereoselective formation of the 1,2-cis (alpha) interglycoside bonds through condensation of the nucleoside derivatives 9 and 10 with 2-(2,3-di-O-benzyl-4-O-stearoyl-beta-L-fucopyranosylsulfonyl) pyrimidine (18). The 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) was prepared by an improved procedure from D-arabinose.     

Evaluation of N-Hydroxy-, N-Metoxy-, and N-Acetoxybenzoyl-Substituted Derivatives of Thymine and Uracil as New Substances for Prevention and Treatment of Long-Term Complications of Diabetes Mellitus

Russian Journal of Bioorganic Chemistry - New uracil and thymine derivatives, N1-,N3- and N1,N3-(RO-benzoyl)-(1H,3H)-pyrimidine- 2,4-diones, were synthesized (RO- is hydroxy, acetoxy- or...     

Radioprotection of pyrimidines by oxygen and sensitization by phosphate: a feature of their electron adducts

The reactions of the electron adducts of thymine, uracil and 1,3-dimethylthymine in the presence of phosphate buffer and low oxygen concentrations have been investigated. Oxygen reacts with the pyrimidine electron adducts and their O(4)-protonated forms to restore the pyrimidine and produce O2-./HO2.. Thus oxygen acts as a radiation protector. In the presence of high buffer concentrations the electron adducts are irreversibly protonated at C(6). On reaction of this radical with oxygen no restitution of the original pyrimidine occurs and the pyrimidine is destroyed. Thus phosphate buffer acts as a radiation sensitizer. Some speculations are made as to the possible relevance of these reactions to biological systems.     

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.     

Polymethylene derivatives of nucleic bases with omega-functional groups. III. N-[7-(2-oxocyclohexyl)-7-oxoheptyl]-substituted pyrimidines

New polymethylene derivatives of nucleic bases containing a beta-dioxo function at the omega-position were synthesized by alkylation of uracil, thymine, and cytosine with 1-(7-chloroheptanoyl)cyclohexan-2-one, and their physicochemical properties were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.