The Ultrasonic Degradation of Thymine1

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The Ultrasonic Degradation of Thymine1 ERIC L. MEAD, RONALD G. SUTHERLAND, AND RONALD E. VERRALL Department of Chemistry and Chemical Engineering, University ofsaskatchewan, Saskatoon, Saskatchewan S7N
The Ultrasonic Degradation of Thymine1 ERIC L. MEAD, RONALD G. SUTHERLAND, AND RONALD E. VERRALL Department of Chemistry and Chemical Engineering, University ofsaskatchewan, Saskatoon, Saskatchewan S7N 0 WO Received March ERIC L. MEAD, RONALD G. SUTHERLAND, and RONALD E. VERRALL. Can. J. Chem. 53,2394 (1975). Sonolysis at 450 khz degrades an aqueous solution of thymine into at least six products of which four, 5-hydroxymethyluracil, cis- and trans-5,6-dihydroxy-5,6-dihydrothymine, and urea have been identified and a fifth tentatively assigned as N-formyl-N'-pyruvylurea. A mechanism of sonolytic degradation is proposed. ERIC L. MEAD, RONALD G. SUTHERLAND et RONALD E. VERRALL. Can. J. Chem. 53,2394 (1975). La sonolyse a 450 khz degrade une solution aqueuse de thymine en au moins six produits dont quatre sont identifies comme &ant I'hydroxy-5 mtthyluracile, le dihydroxy-5,6 dihydro-5,6 thymine cis et trans et I'ur6e; le cinquieme a 6te tentativement identifie i la N-formyl N'- pyruvylurk. On propose un mecanisme de la dkgradation sonolytique. [Traduit par le journal] Chemical reactions that occur in liquids subjected to ultrasonic irradiation (insonation) of sufficient intensity to induce cavitation, have been attributed (1) to a number of processes; the reactive species produced in water and aqueous solutions during cavitation are hydroxyl radicals (2), hydrated electrons (3), hydroperoxyl radicals (4), and hydrogen atoms (5). These species are also produced during the radiolysis of water and in this paper we show that the sonolysis of thymine may be correlated with its radiolytic degradation. The radiolysis of aqueous thymine solutions has been the subject of numerous investigations and studies by Teoule and co-workers (6-11) identified more than 26 products formed during 60Co-y irradiation. Hydroxyl radicals produced by ionizing radiation react with thymine to give 6-hydroxy-5,6-dihydro-5-thyml and 5-hydroxy- 5,6-dihydro-6-thymyl radicals; hydrogen atoms (or e,,- + H,O+) react with thymine to give (5- uracyl)methyl, 5,6-dihydro-5-thyml, and 5,6- dihydro-6-thyml radicals (11). These radicals react. very rapidly with dissolved oxygen (e.g. K(TOH + 0,) = 1.5 x lo9 M -,l S- l (12) to produce hydroperoxide radicals which are reduced to the hydroperoxyhydroxydihydro products (Fig. 1) (5 and 6) and the hydroperoxide products (7, 5-hydroperoxy-5,6-dihydrothymine, cis- and trans-6-hydroperoxy-5,6-dihydrothy- 'For a preliminary communication see ref. 27. mine). The peroxide products are relatively unstable (11) and decompose to give various alcohols (cis- and trans-9, 8, 5-hydroxy-5,6-dihydrothymine, cis- and trans-6-hydroxy-5,6-dihydrothymine). In addition, the hydroxyhydroperoxides decompose to give 5-hydroxy-5- methylbarbituric acid and N-formyl-N'- pyruvylurea and the remaining products result from decomposition of N-formyl-N'-pyruvylurea to give pyruvylurea, urea, and formylurea. El'piner (1) was the first to investigate the effect of ultrasound on purine and pyrimidine bases. He insonated (500 khz) aqueous solutions of the bases at an intensity of 5 W cm-2 in the presence of various gases. In the U.V. spectra of the insonated bases, the absorption maxima were reduced and the minima shifted to longer wavelengths. Uracil was found to be the most sensitive, adenine and guanine less so. In a later study, El'piner and Sokol'skaya (13) insonated aqueous solutions of cytosine, uracil, thymine, and adenine and investigated the products of sonolysis chromatographically but no degradation products were identified. The reduction in intensity of the U.V. absorption band of the various purines and pyrimidines was attributed to ring cleavage (1). Since the effect was considerably reduced in the presence of gases other than oxygen, it was suggested that molecular oxygen played an important role and a smaller oxidative role was assigned to hydroxyl radicals. This study was undertaken to reinvestigate the sonolysis of aerated aqueous thymine solutions MEAD ET AL.: SONOLYSlS OF THYMINE 1 02 reduction 1 and 11.titl.s 8 11 FIG. I. Proposed sonolytic mechanism of degration of aqueous thymine solutions. in light of the mechanism proposed for the radiolysis of thymine. Experimental Ultrasonic irradiation was carried out using a Multisons MC500-1 (Macrosonics Corporation, Cartaret, N.J.) broad band generator coupled to a cobalt barium titanate transducer operated at f 0.6 khz with an average power output of 5 W ~ m based - ~ on calorimetric measurements. The total acoustic power absorbed is not a useful measure of the energy available for chemical reactions since only a very small fraction is used in the formation of free radicals (14). Aliquots (50 ml) of aqueous thymine solutions were insonated in a glass cell equipped with a Mylar 500A plastic window, similar to the design of Spurlock and Reifsneider (15). The temperature inside the cell was kept relatively constant at 25 C by circulating the coupling water, transmitting the acousticenergy to the cell, through a constant temperature (25 f 0.5 OC) bath. The water used in this study was prepared by distillation of previously deionized water from alkaline permanganate. Thymine (Aldrich Chemical Co.) was recrystallized from distilled water. 5-HydroxymethyluraciI (16) and 5,6-dihydroxy-5,6-dihydrothymine (17) were prepared by literature procedures. These procedures give the cis isomer only (18). A mixture of isomers was obtained by refluxing an aqueous solution of the cis isomer at 90 C for 4 h (18). Reagent grade urea was used without further purification. Chromatography was carried out on commercially prepared silica gel G and MN300 cellulose thin-layer (250 pm, 20 x 20 cm) plates and Whatman No. 1 paper. Thymine and derivatives that retain the 5,6-double bond were located using a U.V. lamp with a maximum 2396 CAN. J. CHEM. VOL emission band at 254 nm. The dihydrothymine derivatives urea, and substituted ureas were detected by spraying the chromatograms with 0.5 N aqueous sodium hydroxide, air drying for 30 min, and respraying with a solution ofpdimethylaminobenzaldehyde (PDAB) (1 g in 100 ml 95% ethanol plus 10 ml concentrated hydrochloric acid), after the method of Fink et a/. (19). Iodine vapor was used to detect products on silica gel plates. Ultraviolet spectra of aqueous thymine and insonated thymine solutions were obtained on a Cary 14 recording spectrophotometer using a variable path length cell (model BC14, Research and Industrial Instruments Company, London). The following methods were used in the quantitative analyses of products. The cis and trans isomers of compound 9 were determined by a modification (20) of the method of Roberts and Friedkin (21). The original method determined thymine levels by hypobromination of the heterocyclic ring, followed by base hydrolysis to give the glycol. Continued base hydrolysis of the glycol yielded acetol which was condensed with o-aminobenzaldehyde to form 3-hydroxyquinaldine. Elimination of the hypobromination step in this work permitted the quantitation of thymine glycol in the presence of thymine. The fluorescence was measured at 440 nm (excitation at 310 nm) in a 1.0 cm quartz cell using an Aminco-Bowman spectrophotofluorimeter. Total peroxides were measured as hydrogen peroxide using the molybdate catalyzed oxidation of iodide to the triiodide anion (22) which was measured spectrophotometrically at 350 nm in a 1.0 cm quartz cell. Hydrogen peroxide was determined spectrophotometrically following the method of Schumb et a/. (23) using acidic titanium sulfate. Although not as sensitive as the triiodide anion method, it is specific for hydrogen peroxide in the presence of organic peroxides (24). Both methods for the determination of peroxide obeyed Beer's law in the concentration range 0-1 x M. Urea was determined spectrophotometrically by measuring the visible absorption at 420 nm arising from the reaction of urea with p-dimethylaminobenzaldehyde (25). Results Aqueous thymine solutions (2 x to 2 x M) showed a linear decrease with time in the U.V. absorption maximum at 264 nm during insonation at constant ultrasonic intensity. The characteristic changes observed were reduction of the band at 264 nm; reduction and shift to longer wavelengths of the minimum; increase and shift to shorter wavelengths of the band at 204 nm. Semiquantitative data on the rate of degradation of thymine and relative yields of products were obtained. Figure 2 shows that the degradation rate, measured spectrophotometrically for three different initial concentrations, was constant over at least a 2-h insonation period. However, one of the products (8) (Fig. 1) has a molar extinction coefficient (E 8.00 x lo3 M-' Time (h) FIG. 2. Rate of degradation of aqueous thymine solutions at khz. cm- l, A, 261 nm, 0.01 N HCl(16)) comparable to that of thymine and contributes to the measured absorption at 264 nm 'as the degradation proceeds. Attempts to determine the yield of 8 and unreacted thymine by spectrophotometric measurements of solutions, obtained by eluting the separated compounds from chromatograms, did not give accurate data. Table 1 contains some data on the product yields for two initial thymine concentrations. The methods used for the analysis were previously outlined in the experimental section. The products were separated and identified by cochromatography with synthetic samples and by their reaction to spraying with p-dimethylaminobenzaldehyde. The products separated and positively identified are cis- and trans-5,6- dihydroxy-5,6-dihydrothymine (thymine glycol), 5-hydroxymethyluracil, and urea. At least two more products, one tentatively identified as N- pyruvyl-n'-formylurea and the other unidentified, have also been isolated. Table 2 tabulates the R, values measured for each of the above products on a variety of stationary and mobile phases. The formation of 5-hydroxymethyluracil during sonolysis was verified by cochromatography with synthetic samples. As well, samples were MEAD ET AL.: SONOLYSIS OF THYMINE 2397 TABLE 1. Product yields* from two initial thymine concentrations Thymine Total Hydrogen Thymine Time Thymine glycol peroxide peroxide Urea decomposed (h) (M lo3) (M x lo3) (M x lo3) (M x lo3) (M x lo3) (M x lo3) O *Arithmetic average of at least two determinations for each product. eluted from developed chromatograms for spectral comparison by the method of Cline et al. (1 6). The ratio of the absorbances at and were calculated; for the eluted sample, the ratios were = 0.40 and = 0.74, whereas for the synthetic mixture the ratios were 0.38 and 0.77, respectively, in methanol. The cis and trans isomeric glycols were identified by cochromatography and by their reaction to spraying with PDAB after base hydrolysis, which gave a blue-green color for each. Urea was also identified by cochromatography and by the bright lemon color obtained by spraying the chromatogram with PDAB without prior base hydrolysis. The product T.l is thought to probably be N- pyruvyl-n'-formylurea due to its color reaction (yellow-red) after spraying with PDAB after base hydrolysis while the Rf values for this product on systems 11 and VI were similar to those reported by Teoule and Cadet (6). The product T.2 has not yet been identified. Discussion The results of this work have shown that sonolysis of aqueous thymine solutions in the concentration range 2 x to 2 x lo-' M gives rise to products identical to those produced by radiolysis. The degradation of thymine was found to vary linearly with insonation time with a small dependence on initial concentration as was observed for the radiolysis of thymine (6). Analysis of the initial rate data shown in Fig. 2 was carried out for all but the highest initial concentration of thymine. If the initial rate of reaction, r, is related to the reactant concentration, c, by the relation r = k [c] , then a value of the order of reaction, n, can be calculated from two initial rate conditions. The value obtained was 0.1 f 0.15 and within experimental error the decomposition follows apparent zero-order kinetics. The large error in n includes some estimate of error arising out of the interference by product 8 in the spectrophotometric determination of thymine. A value of 1.8 f 0.3 x M min-' was obtained for the zero-order rate constant. The data in Table 1 are insufficient to carry out any meaningful kinetic analysis. However, some general observations can be made. After one hour of insonation approximately the same amounts of the thymine glycols, cis- and trans-9, were found for two different intial thymine concentrations. The method used in this study to determine the amounts of 9 appeared to be free from interference from thymine on monohydroxythymine derivatives. The initial yields of hydrogen peroxide are in agreement with those previously reported (2, 15) for air-saturated water insonated at approximately the same frequency. An estimate of the organic peroxide concentration was obtained from the difference between total peroxide and hydrogen peroxide concentrations. The organic peroxide precursors of the stable products of thymine degradation appear to never form more than ca. 5% of the total amount of thymine decomposed. Teoule and Cadet (6) found that at a high dose of 60Co-y irradiation, the total peroxide radiochemical yield G(peroxide) = 0.5 as compared to G(-T) = The amounts of urea formed during sonolysis of aqueous thymine solutions did not exceed 20%, of the amount of decomposed thymine and appeared to decrease with increasing insonation time. This small amount of urea formed is in agreement with the radiochemical yield of urea, TABLE 2. Chromatographic R, values of products on a variety of stationary and mobile phases Stationary Mobile 5-Hydroxymethyl- trans-5,6-dihydroxy- cis-5,6-dihydroxyphase* phase? Thymine Urea uracil 5,6-dihydrothymine 5,6-dihydrothymine T'. 1 0 2: T.2 + I I I I IV r y V VI I S VII VIII 'Stationary phases: I 11: silica gel G. iii 1V: MN 300 cellulose: V-VII: Whatman No. I paper. thlobilephases: I, acktonitrile - phosbhaie buffer (0.2 M Na2HP0, and 0.2 MNaH2P04) (85: 15, vlv); 2, ethyl acetate - isopropyl alcohol - water (75: 16:9); 3, n-propyl alcohol - water (10: 3); 4, n-butvl alcohol -methanol - water - ammonium hvdroxide (60:20:20: 1): 5. n-butvl alcohol-water (86: 14); 6, tert-butyl alcohol -methyl ethyl ketone - water - formic acid (40: 30: 15: 15); 7, tertbutyl-alcohol - methyl ethyl ketone - water - ammonium hydroxide (40: 30: 15: 15j. All proportions of the mobile phases are by volume. LA MEAD ET AL.: SONOLYSIS OF THYMINE 2399 G(urea) = (10). The decrease in urea concentration with increasing insonation time may be accounted for in this work by further reaction of urea with nitrous acid, which is a product of the insonation of air-saturated water (26). Although the amount of 8 could not be measured quantitatively it was possible, by visual comparison of the fluorescence intensity under U.V. illumination of chromatographic spots from aliquots of standard solution and insonated solutions, to estimate the maximum concentration of 8 to be 1 x M. From the results obtained in this work it appears that the radiolysis mechanism of thymine destruction proposed by Teoule et al. (10) may be adapted to the sonolytic degradation of thymine (Fig. I). Cavitation produces the reactive species.oh, H-, and e,,- ; all of the sonolytic products may be accounted for by reaction of hydroxyl radicals and/or hydrogen atoms with thymine. Hydroxyl radicals undergo additional reactions with thymine to produce 6-hydroxy-5,6- dihydro-5-thymyl (2) and 5-hydroxy-5,6-dihydro-6-thyml (3) radicals; hydrogen atoms (or hydroxyl radicals) undergo abstraction reactions with thymine to give (5-uracy1)methyl radicals (4). These radicals react very rapidly with dissolved oxygen to produce the hydroperoxy radicals which are reduced to the hydroperoxide products, 5, 6, and 7. The hydroperoxide products decompose to 5-hydroxymethyluracil (8) and cis- and trans-5,6-dihydroxy-5,6-dihydrothymine (9). In addition, the cis- and trans-5 (or 6)hydroperoxy-6(or 5)-dihydrothymines (5 and 6) probably undergo ring opening to give N-formy1-N'- pyruvylurea (10) which decomposes to urea (11). The above mechanism accounts for all of the identified products in this study. 2. A. WEISSLER. J. Am. Chern. Soc. 81, 1077 (1959). M. ANBAR and I. PECHT. J. Phys. Chern. 68, 352 (1964). M. HAISSINSKY and R. KLEIN. J. Chern. Phys. Physico-chirn. Biol. 65, 326 (1968). 3. M. A. MARGULIS and A.N. MAL'TSEV. RUSS. J. Phys. Chern. 42, 1412 (1968).(English translation of Zh. Fiz. Khirn. 42,2660 (1x8)). 4. B. LIPPITT, J. M. MCCORD, and I. FRIDOVICH. J. Biol. Chern. 247,4688'(1972). 5. R. RIVAYRAND and M. HAISSINSKY. J. Chirn. Phys. 59, 623 (1962). M. ANBAR and I. PECHT. J. Phys. Chern. 68, 1460 (1968). 6. R. TEOULE and J. CADET. Bull. Soc. Chirn. Fr. 927 (1970). 7. J. CADET and R. TEOULE. Int. J. Appl. Radiat. Isot. 22,273 (1971). 8. R. TEOULE, J. CADET, and J. ULRICH. C.R. Ser. C, 270,362 (1970). 9. R. TEOULE and J. CADET. Chern. Cornrnun (1971). 10. R. TEOULE, J. CADET, M. POLVERELLI, and A. CIER. Peaceful uses of atomic energy. Vol. 13. U.N., New York. Intl. At. En. Agency, Vienna J. CADET and R. TEOULE. Biochirn. Biophys... Acta, 238,8 (1971) H. LOMAN and M. EBERT. Int. J. Radiat. Biol ~ (1970). 13. I. E. EL'PINER~~~ A. V. SOKOL'SKAYA. Dokl. Akad. Nauk S.S.S.R. 153,200 (1963). 14. M. A. MARGULIS. SOV. Phys Acoust. 15,135 (1969). 15. L. A. SPURLOCK and S. B. REIFSNEIDER. J. Am. Chern. Soc. 92,6112 (1970). 16. R. E. CLINE, R. M. FINK,^^^ K. FINK. J. Am. Chern. SOC. 81,2521 (1959) BAUDISCH and D. DAVIDSON. J. Biol. Chern. 64, 233 (1925). 18. J. ULRICH, R. TEOULE, R. MASSOT, and A. CORNU. Org. Mass Spectrorn. 2, 1183 (1969). 19. R. M. FINK, R. E. CLINE, C. MCGAUGHEY, and K. FINK. Anal. Chern. 28,4(1956). 20. M. DANIELS and M. GRIMISON. Biochirn. Biophys. Acta, 142,293 (1967). 21. D.Ros~~~sandM.F~1~~~1~.J.Biol.Chem.233,483 (1958). 22. A. 0. ALLEN, C. J. HOCHANADEL, J. A. GHORMLEY, and T. W. DAVIS. J. Phys. Chern. 56,575 (1952). 23. W. C. SCHUMB, C. N. SATTERFIELD, and R. L. WENTWORTH. Hydrogen peroxide. Reinhold Publish- The authors acknowledge financial support of this work ing Corp., New York, N.Y from the Research Corporation and the National Re- 24. G. NETTESHEIM. 2. Anal. Chern. 191,45 (1962). search Council of Canada. 25. G. W. WATT and J. D. CHRISP. Anal. Chern. 26, 452 ( 1954). 26. E. L. MEAD, R. G. SUTHERLAND, and R. E. VER- 1. I. E. EL'PINER. Ultrasound: physical, chemical and RALL. TO be published. biological effects. Consultants Bureau, New York, 27. E. L. MEAD, R. G. SUTHERLAND, and R. E. VER- N.Y RALL. Chern. Cornrnun. 414 (1973).
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