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Isolation and identification of the irradiation product of thymine
55 °
PRELIMINARY NOTES
The U.S. Public Health Service supported this work with a grant (No. A-3464) to S.C. and a Traineeship to J.B.
Department of Biochemistry, University of California, Davis, California (U.S.A .)
JOHN BAKER STERLING CHAYK1N
1 j. M. SHI~WAN, Biochem. Soc. Symposia, 6 (1951) 28. 2 H. MULLER AND I. IMMENDORFER, Z. Physiol. Chem., 275 (1942) 267. s W. LINTZEL, Biochem. Z., 273 (1934) 243. 4 E. IR. NORRIS AND G. J. BENOIT, J. Biol. Chem., 158 (1945) 443. 5 I{. L. A. TARR, J. Fisheries Research Board Can., 5 (1941) 211. s I. D. FRANTZ AND N. L. IR. BUCHgR, J. Biol. Chem., 206 (1954) 471. 7 SOMMELET AND FERRAND, Bull. soc. ehim. France, 25 (1919) 457s T. T. TCHEN AND I~2. ]3LOCH, J. Biol. Chem., 226 (1957) 921. 9 H. S. MASON, Advances in En,.ymol., 19 (1957) 79. 10 t-I. 1~. D. JAcogs AND W. S. ttOFFMAN, J. Biol. Chem., 93 (1931) 685. 11 S. CHAYKIN AND t(. ]~LOCH, Biochim. Biophys. Acta, 31 (1959) 213. is M. S. FISH, N. M. JOHNSON, E. P. LAWRENCE AND E. C. HORNING, Biochim. Biophys. Acla, 18 (1955) 564 • 13 A. G. C,-ORNALL, C. J. BARDAWILL AND Y][. M. DAVID, J. Biol. Chem., 177 (1949) 751.
Received May 7th, 196o Biochim. Biophys. Acta, 41 (196o) 548-55 °
Isolation and identification of the irradiation product of thymine We have described the rapid conversion b y u.v. radiation of thymine in a frozen aqueous solution into a low-absorbing substance 1. This transfer is reversible since renewed irradiation of the thawed solution of the reaction product leads again to thymine. The irradiation product of thymine has now been isolated and a suggestion for the structure can be made. Since thymine shows a very high molecular extinction, only dilute solutions can be used in the irradiation procedure (4o0 mg/1). After freezing in large Petri-discs (35-ml portions) the thymine solution was subjected to u.v. (2537 A). The solutions were allowed to thaw and the irradiation product was freed from thymine b y a repeated extraction with absolute ethanol. In contrast to thymine, the product is almost insoluble in this solvent. The completeness of the extraction was determined by measuring the extinction of the extract. After complete removal of thymine (no residual absorption at 264 m~) the residue was recrystallized from water (small needles, decomp. 32o°). The elementary analysis agrees completely with that of thymine. (Found: C, 47-57, 47.60; H, 4.94, 4-81; N, 22.21, 22.09. Calc. for CsHeO2N2: C, 47.62; H, 4.8o; N, 22.21). The substance therefore must be an isomer or a polymer of thymine. Molecular-weight determination by the usual methods was impossible on account of the low solubility in organic solvents and in camphor and similar substances. For this reason the method of SIGNER (isothermic distillation) ~ was used. The apparatus was made according to CLARK 3. In order to obtain a sufficiently high vapour pressure of the solvent (water), the temperature of the surrounding water bath was kept at 85 ° . Even at this temperature the concentration could not be higher than o.oi M. From the results obtained in this way we m a y conclude upon a dimer. This assumption has been supported by a crystallographic investigation. The Biochim. Biophys. Acta, 41 (196o) 55o-551
PRELIMINARY NOTES
551
irradiation product crystallizes in the rhombic system and the elementary cell contains 16 molecules of a monomer size. Since this number in a rhombic crystal is usually 8, a dimer is probable. A third indication for a dimeric structure is found in the formation of a t h y m i n e uracil addition product upon irradiation of a mixture of thymine and uracil in frozen solution. From mass-spectrographic results, a molecular weight of 126 (monomer) was found. I t may, however, be assumed that the molecule is very susceptible to the electron bombardment to which it is subjected in the method and will be immediately split in two thymine molecules. As already mentioned above, the same results can also be obtained by u.v. irradiation of a solution of the irradiation product in water 4. The i.r. spectrum contains the small peaks of a cyclobutane ring system (about 870 cm-1). In view of the high chemical stability, a C-C bond formation is plausible. The absence of an u.v. absorption in the neighbourhood of 260 m/~ and the improbability of other possibilities suggest the following structure O
H
II .C
H-N/ 1
O:C~
I N
Me H
o I
I
l
/ ~C~--~C. --N--H N/ H Me ~ C / I
H
II
O
Similar dimerisations are known in the literature 5. The other dimer with both methyl groups at adjacent C atoms is less probable on account of nuclear-magneticresonance analysis. No spin-spin coupling of the two neighbouring H atoms at the cyclobutane ring can be observed. This does not mean that we will rule out or deny the existence of a dimer of this structure. Under special conditions (e.g. in deoxyribonucleic acid) its formation might be possible. This investigation was supported by the U.S. Army. We wish to express our gratitude to Dr. J. BOLDINGH, director of the Unilever Research Laboratory, Vlaardingen, for giving us the opportunity to have the nuclear magnetic resonance and mass-spectrographic analyses done. These analyses were performed by Ir. R. KEUNING and Ir. W. SONNEVELD,whom we thank for their great interest and help. We should also like to thank Ir. J. H. PALM for the crystallographic investigation and Mr. M VAN LEEUWEN for the elementary analysis.
Biochemical and Biophysical Laboratory of the Technological University R. BEUKERS of Delft, Julianalaan 67 (The Netherlands) W. BERENOS 1 R. R. 3 E. 4 R. 5 A.
BEUKERS, J. IJLSTRA AND W. BERENDS, Rec. tray. chim., 77 (1958) 729 . SmN~R, An*l., 478 (193 o) 246. P. CLARK,Anal. Chem., 13 (1941) 820. BEUKERS, J. IJLSTRA AND W. BERENOS, Rec. tray. chim., 78 (1959) 883. SCH6NBERG, Praparative organische Photochemie, Springer Verlag, Berlin, 1958.
Received May 27th, 196o Biochim. Biophys. Acta, 41 (196o) 550-551
PRELIMINARY NOTES
The U.S. Public Health Service supported this work with a grant (No. A-3464) to S.C. and a Traineeship to J.B.
Department of Biochemistry, University of California, Davis, California (U.S.A .)
JOHN BAKER STERLING CHAYK1N
1 j. M. SHI~WAN, Biochem. Soc. Symposia, 6 (1951) 28. 2 H. MULLER AND I. IMMENDORFER, Z. Physiol. Chem., 275 (1942) 267. s W. LINTZEL, Biochem. Z., 273 (1934) 243. 4 E. IR. NORRIS AND G. J. BENOIT, J. Biol. Chem., 158 (1945) 443. 5 I{. L. A. TARR, J. Fisheries Research Board Can., 5 (1941) 211. s I. D. FRANTZ AND N. L. IR. BUCHgR, J. Biol. Chem., 206 (1954) 471. 7 SOMMELET AND FERRAND, Bull. soc. ehim. France, 25 (1919) 457s T. T. TCHEN AND I~2. ]3LOCH, J. Biol. Chem., 226 (1957) 921. 9 H. S. MASON, Advances in En,.ymol., 19 (1957) 79. 10 t-I. 1~. D. JAcogs AND W. S. ttOFFMAN, J. Biol. Chem., 93 (1931) 685. 11 S. CHAYKIN AND t(. ]~LOCH, Biochim. Biophys. Acta, 31 (1959) 213. is M. S. FISH, N. M. JOHNSON, E. P. LAWRENCE AND E. C. HORNING, Biochim. Biophys. Acla, 18 (1955) 564 • 13 A. G. C,-ORNALL, C. J. BARDAWILL AND Y][. M. DAVID, J. Biol. Chem., 177 (1949) 751.
Received May 7th, 196o Biochim. Biophys. Acta, 41 (196o) 548-55 °
Isolation and identification of the irradiation product of thymine We have described the rapid conversion b y u.v. radiation of thymine in a frozen aqueous solution into a low-absorbing substance 1. This transfer is reversible since renewed irradiation of the thawed solution of the reaction product leads again to thymine. The irradiation product of thymine has now been isolated and a suggestion for the structure can be made. Since thymine shows a very high molecular extinction, only dilute solutions can be used in the irradiation procedure (4o0 mg/1). After freezing in large Petri-discs (35-ml portions) the thymine solution was subjected to u.v. (2537 A). The solutions were allowed to thaw and the irradiation product was freed from thymine b y a repeated extraction with absolute ethanol. In contrast to thymine, the product is almost insoluble in this solvent. The completeness of the extraction was determined by measuring the extinction of the extract. After complete removal of thymine (no residual absorption at 264 m~) the residue was recrystallized from water (small needles, decomp. 32o°). The elementary analysis agrees completely with that of thymine. (Found: C, 47-57, 47.60; H, 4.94, 4-81; N, 22.21, 22.09. Calc. for CsHeO2N2: C, 47.62; H, 4.8o; N, 22.21). The substance therefore must be an isomer or a polymer of thymine. Molecular-weight determination by the usual methods was impossible on account of the low solubility in organic solvents and in camphor and similar substances. For this reason the method of SIGNER (isothermic distillation) ~ was used. The apparatus was made according to CLARK 3. In order to obtain a sufficiently high vapour pressure of the solvent (water), the temperature of the surrounding water bath was kept at 85 ° . Even at this temperature the concentration could not be higher than o.oi M. From the results obtained in this way we m a y conclude upon a dimer. This assumption has been supported by a crystallographic investigation. The Biochim. Biophys. Acta, 41 (196o) 55o-551
PRELIMINARY NOTES
551
irradiation product crystallizes in the rhombic system and the elementary cell contains 16 molecules of a monomer size. Since this number in a rhombic crystal is usually 8, a dimer is probable. A third indication for a dimeric structure is found in the formation of a t h y m i n e uracil addition product upon irradiation of a mixture of thymine and uracil in frozen solution. From mass-spectrographic results, a molecular weight of 126 (monomer) was found. I t may, however, be assumed that the molecule is very susceptible to the electron bombardment to which it is subjected in the method and will be immediately split in two thymine molecules. As already mentioned above, the same results can also be obtained by u.v. irradiation of a solution of the irradiation product in water 4. The i.r. spectrum contains the small peaks of a cyclobutane ring system (about 870 cm-1). In view of the high chemical stability, a C-C bond formation is plausible. The absence of an u.v. absorption in the neighbourhood of 260 m/~ and the improbability of other possibilities suggest the following structure O
H
II .C
H-N/ 1
O:C~
I N
Me H
o I
I
l
/ ~C~--~C. --N--H N/ H Me ~ C / I
H
II
O
Similar dimerisations are known in the literature 5. The other dimer with both methyl groups at adjacent C atoms is less probable on account of nuclear-magneticresonance analysis. No spin-spin coupling of the two neighbouring H atoms at the cyclobutane ring can be observed. This does not mean that we will rule out or deny the existence of a dimer of this structure. Under special conditions (e.g. in deoxyribonucleic acid) its formation might be possible. This investigation was supported by the U.S. Army. We wish to express our gratitude to Dr. J. BOLDINGH, director of the Unilever Research Laboratory, Vlaardingen, for giving us the opportunity to have the nuclear magnetic resonance and mass-spectrographic analyses done. These analyses were performed by Ir. R. KEUNING and Ir. W. SONNEVELD,whom we thank for their great interest and help. We should also like to thank Ir. J. H. PALM for the crystallographic investigation and Mr. M VAN LEEUWEN for the elementary analysis.
Biochemical and Biophysical Laboratory of the Technological University R. BEUKERS of Delft, Julianalaan 67 (The Netherlands) W. BERENOS 1 R. R. 3 E. 4 R. 5 A.
BEUKERS, J. IJLSTRA AND W. BERENDS, Rec. tray. chim., 77 (1958) 729 . SmN~R, An*l., 478 (193 o) 246. P. CLARK,Anal. Chem., 13 (1941) 820. BEUKERS, J. IJLSTRA AND W. BERENOS, Rec. tray. chim., 78 (1959) 883. SCH6NBERG, Praparative organische Photochemie, Springer Verlag, Berlin, 1958.
Received May 27th, 196o Biochim. Biophys. Acta, 41 (196o) 550-551