BIOCHEMISTRY
Photochemistry of 5-Bromo-1,3-dimethyluraciI in Aqueous Solution" Hiroshi Ishiharat and Shih Yi Wang
ABSTRACT: 5,5 '-Di-l,3-dimethyluracil (IV), 1,3-dimethyluracil (111), sym-dimethyloxamide (11), symdimethylurea (VI), methylamine, ammonia, 5-carboxy1,3-dimethyluracil (VII), and acetic acid formed by ultraviolet irradiation (mainly 254 mp) of 5-bromo1,3-dirnethyluracil in aqueous solution were quantitatively isolated and identified. Based on the isolation of the first two photoproducts, a free radical reaction
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acteriophages (Stahl et al., 1961;Sauerbier, 1961), bacterial (Greer, 1960; Kaplan et al., 1962) and mammalian cells (Djordjevic and Szybalski, 1960) containing 5-bromouracil or 5-iodouracil in place of thymine in their deoxyribonucleic acid (DNA) are much more sensitive to irradiation than the phages and cells with normal DNA. p h e references cited in a previous paper (Ishihara and Wang, 1966) are pertinent to this work.] The mechanism by which this occurs remains unknown. The irradiation sensitization produced by these 5-halogenouracils could be due either to their greater chemical reactivity or to the inability of existing cellular mechanisms to repair the type of damage (irradiation product) produced. In either case, the isolation and identification of the products of halogenated uracils and the study of their chemical mechanisms is essential to a complete understanding of the enhanced irradiation sensitivity. The photochemical behavior of methylpyrimidines is quite similar to that of their analogs in nucleic acids. Hence, methylpyrimidines have been successfully used as model compounds for chemical studies related to photobiology (Moore and Thomson, 1955; Wang et al., 1956; Wang, 1959a). These chemical studies might also yield information about the effect of methyl groups on the photochemical reactions of pyrimidines. In this study, 5-bromo-l,3-dimethyluracil (I)' was used as the model compound. It is hoped that this study will assist
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* F r o m the Departments of Radiological Science and Biochemistry, The Johns Hopkins University School of Hygiene and Public Health, Baltimore. Maryland 21205. Receiued Janua r y 6, 1966; revised April 11, 1966. This research has been supported in part by a Contract AT(30- 1)-2798 from the U. S. Atomic Energy Commission and a Research Career Development Award from the Division of General Medical Science, U. S. Public Health Service. t On leave of absence from Chemical Laboratory, Nagoya City University, Mizuho-ku, Nagoya, Japan.
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ISHIHARA
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Y1 WANG
mechanism was suggested for this photochemical reaction. The homolysis of the C-Br bond to form uracil radicals and bromine atoms may be responsible for the enhanced irradiation sensitization of bromouracildeoxyribanucleic acid (BU-DNA) in vivo, and the coupled product, 5,s '-di-l,3-dimethyluracil, may serve as a model for the study of radiation and photobiology.
our further work with 5-bromouracil, the nucleoside, and the nucleotide. In a previous paper (Ishihara and Wang, 1966), we have reported the isolation and identification of symdimethyloxamide (11), 1,3-dimethyluracil (111), and $ 5 '-di-l ,3-dimethyluracil (IV) from the irradiation of BDMU. The probable mechanism for the formation of DMU and DMU-DMU through common DMU radicals (V) was considered. The possible importance of the free-radical mechanism and the formation of coupled products, such as DMU-DMU, in irradiation and photobiology was discussed. The present paper deals with further studies on the photochemistry of DMU in aqueous medium, including the quantitative determination of five other products which were not reported in the preceding paper. Experimental Procedures Melting points were determined on a Fisher-Johns block and are uncorrected. Infrared spectra were measured on a Perkin-Elmer Model 21 recording spectrophotometer. Ultraviolet spectra were measured on a Beckman Model DK-1 recording spectrophotometer. Microanalyses were performed by Mr. J. Walter at the Johns Hopkins University. Paper chromatography was carried ovt on Whatman No. 1 filter paper unless otherwise stated and the ratios given for the eluents are by volume. Evaporation of the solvents was carried out by a rotary evaporator below 40'. The irradiation apparatus has been described previously (Wang, 1958). General Electric germicidal
1 Abbreviations used in this work: BDMU, 5-bromo-1,3dimethyluracil ; DMU, 1,3-dimethyluracil; DMU-DMU, 5,s 'di-1,3-dimethyluracil; CDMU, 5-carboxy-l,3-dimethyluracil; OH-DMU, S-hydroxy-l,3-dimethyluracil;DMU.,1,3-dimethyluracil radical.
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tubes (G15T8) which emit mainly 254-mp wavelength light were used as light source. Chemically pure samples (BDMU I) prepared according to the method of Wang (1959b) were used [X:i' 241 mp (E 1.76 X 103). 283 mp (e 8.66 X lo3), mp I 84-1 85 "1. Irradiation of BDM U and Partial Fractionation of the Irradiation Mixture An aqueous solution of BDMU (1 mM, 10 1.) was irradiated for 1.5 hr at room temperature. During irradiation, the absorbancy of the solution at 283 mp decreased to ca. 45z of the original and the pH decreased from 6.0 to ca. 3.0. The irradiated solution was adjusted to pH 9.0 with 0.1 N NaOH solution, and was concentrated to 10 ml. The distillate, or distillate fraction, was collected in a condenser containing 10 ml of 4 N HCl. The solution concentrated to 10 ml as above was extracted continuously with ether for 5 days. The ether fraction obtained was kept for the analysis of neutral photoproducts. The aqueous solution that remained after extraction with ether was evaporated to dryness. The residue, aqueous fraction, was retained for identification of the acidic photoproducts. Isolation and ldenti~cationof Photoproducts Distillate Fraction. A. QUALITATIVE IDENTIFICATION OF THE VOLATILE BASES. The distillate fraction was concentrated to a small volume and chromatographed in solvent systems A [I-butanol-acetic acid-water (4: 1 :5 ) ] and B [1-butanol-acetic acid-water (2 :1 :l)] by descending technique. The dried chromatograms were sprayed with ninhydrin (0.1% in acetone). Heating revealed two spots in both systems: a violet spot (RF 0.38 in system A, 0.60 in B) and a yellow one (RF0.23 in A, 0.46 in B) which coincided well with parallel runs using authentic samples of methylamhe hydrochloride (violet) and ammonium chloride (yellow), respectively (Alcfintara and Wang, 1965). Their identification was established by comparing the infrared spectra of the eluted substances from the chromatograms with authentic samples of methylamine hydrochloride and ammonium chloride. B. QUANTITATIVE DETERMINATION OF THE TOTAL VOLATIVE BASES. An aqueous solution of BDMU (1 rnM, 1 1.) was irradiated for 1.5 hr. The irradiated solution was acidified with 1 ml of concentrated HCI and was then evaporated to dryness. The residue was dissolved in 10 ml of water and aliquots of 3 ml were used for the determination of the total volatile bases according to the modified method of Archibald (1943) and Varner et al. (1953). After 20 min of distillation, 2 ml of ethanol was added to the sample chamber. Distillation was continued for 15 min and the content in the receiver was then titrated to pH 5 with 0.01 N HCI. This process was repeated until the content in the receiver showed a pH no greater than 6 (addition of
