The reactions of thymine and thymidine with ozone

Abstract

The ozonolysis of thymine and thymidine has been investigated by a product study complemented by kinetic studies using spectrophotometry, conductometry and stopped-flow with optical and conductometric detection. Material balance has been obtained. Ozonolysis of thymine (k = 3.4 x 10(4) dm(3) mol(-1) s(-1)) leads to the formation of the acidic (pK(a) = 4) hydroperoxide 1-hydroperoxymethylene-3-(2-oxopropanoyl)urea 5 (similar to34%), neutral hydroperoxides (possibly mainly 1- hydroperoxyhydroxymethyl-3-(2-oxopropanoyl)urea 6, total similar to41%) and H2O2 (25%, with corresponding formation of 1-formyl-5-hydroxy-5-methylhydantoin 11). The organic hydroperoxides decay (similar to1.1 x 10(-3) s(-1) at 20 degreesC, 1.3 x 10(-4) s(-1) at 3 degreesC) releasing formic acid (formation of 5-hydroperoxy-5-methylhydantoin 18) and also to some extent H2O2 (and 11). After 100 min, the formic acid yield is 75%. Upon treatment at high pH, it increases to 100%. Reduction of the organic hydroperoxides with bis(2- hydroxyethyl)sulfide (k = 50 dm(3) mol(-1) s(-1)) leads to 11 whose subsequent treatment with base yields 5-hydroxy-5- methylhydantoin 13 in 100% yield. It is suggested that the Criegee ozonide formed upon reaction with ozone at the C(5)- C(6) double bond opens heterolytically in two directions with subsequent opening of the C(5)-C( 6) bond. In the preferred route (75%), the positive charge resides at C(6). Deprotonation at N(1) gives rise to 5, while its reaction with water yields 6. Loss of formic acid yields 5-hydroperoxy-5- methylhydantoin 18. Reduction of 5 and 6 with the sulfide yields 11. In the minor route (25%), the positive charge remains at C(5) followed by a reaction with water. The resulting alpha-hydroxy hydroperoxide rapidly loses H2O2 (formation of 11). In basic solution, singlet dioxygen is formed (8%). The concomitant product, 5,6-dihydroxy-5,6-dihydrothymine has been detected. In the ozonolysis of thymidine, the rapid formation of conductance (k = 0.55 s(-1)) is due to the release of acetic acid (18%). In this reaction a short-lived hydroperoxide is destroyed. As a consequence of this, 25 s after ozonolysis the total hydroperoxide yield is only 78% (including 8% H2O2). The products corresponding to acetic acid are suggested to be CO2 and N-(2-deoxy-beta-D-erythropentofuranosyl)formylurea 22. A number of organic hydroperoxides have been detected by HPLC by post-column derivatisation with iodide. An acidic hydroperoxide such as 5 in the case of thymine is not among the products. Upon sulfide reduction, the organic hydroperoxides yield mainly (43-50%) N-1-(2-deoxy-beta-D-erythropentofuranosyl)-5-hydroxy- 5-methylhydantoin 23. The reasons for some striking differences in the ozonolyses of thymine and thymidine are discussed

DNA degradation with ozone

“…One of the most recent works on DNA degradation with ozone [8] has already reported the secondorder rate degradation rate constant under neutral conditions: 410 L mol −1 s −1 . The work of Von Sonntag and coworkers [8,9] has shown that the ozonization reaction of the free purine and pyrimidine bases depend strongly on their protonation state and hence from the pH of the reaction medium. The reaction velocity increases by several orders of magnitude under alkaline conditions in comparison to acidic conditions [8].…”

“…However, the collapse of the DNA supramolecular structure is even more pronounced in comparison to RNA. The second-order rate constants concerning the reaction between the nucleobases and ozone available from literature [8,9] suggest that the most reactive site in the DNA molecule is offered by the thymine base (rate constant 3.4 × 10 4 L mol −1 s −1 ) followed by guanine base (rate constant 1.6 × 10 4 L mol −1 s −1 ). Cytosine is one order of magnitude less reactive than the former two bases and adenine is even lessreactive three orders of magnitude less than the former two.…”

“…The natural continuation of our studies on ozone degradation of biological macromolecules [2][3][4][5][6][7] involves now the DNA macromolecule. As pointed out by other authors [8,9] the studies on the action of ozone on DNA and RNA molecules have important implications in the disinfection mechanism exerted by ozone in drinking water treatment or even air disinfection in closed ambient. In fact both bacteria and viruses result damaged and inactivated because the oxidizing action of ozone is directed toward the nucleic acids of these organisms [8].…”

Ozone Degradation of Biological Macromolecules: Proteins, Hemoglobin, RNA, and DNA

“…Much more attention has been dedicated to the ozone degradation of nucleotides and nucleosides (Theruvathu et al, 2001;Flyunt et al, 2002). The ozone degradation of RNA has important implications on the inactivation and destruction of viruses and bacteria by the ozone disinfection and water and wastewater treatment and in the understanding of the mechanism of cell damage in vivo.…”

Evaluation of ozone for preventing fungal influenced corrosion of reinforced concrete bridges over the River Nile, Egypt

“…Microbial cell damaged and inactivated because the oxidizing action of ozone is directed toward the nucleic acids of these organisms (Flyunt et al 2002). The initial ozone attack is directed against the cell walls with subsequent leakage of cellular content and destruction of the bound DNA, however, ozone is also mutagen and this implies the direct DNA damage (Theruvathu et al 2001).…”