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The permanganate oxidation of thymine
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96524
T H E PERMANGANATE OXIDATION OF THYMINE* S H I G E R U I I D A AND H I K O Y A H A Y A T S U
Faculty o/ Pharmaceutical Sciences, University oI Tokyo; Bunkyo-ku, Tokyo (Japan) (Received J a n u a r y i6th, 197 o)
SUMMARY
I. Mild permanganate oxidation of thymine yielded cis-thymine glycol (cis5,6-dihydroxy-5,6-dihydrothymine ) and 5-hydroxy-5-methylbarbituric acid. Under certain reaction conditions, only these two compounds were the products of the reaction. 2. While cis-thymine glycol was resistant to hydrolysis at neutral pH, 5-hydroxy-5-methylbarbituric acid readily underwent hydrolysis to give methyltartronylurea. 3. In this oxidation reaction, 5-hydroxy-5-methylbarbituric acid, which is higher in the oxidation level than thymine glycol, is formed not via the thymine glycol but directly from thymine. 4. When the oxidation of thymine was carried out at an acidic pH, 5-hydroxy5-methylbarbituric acid was predominantly produced, whereas thymine glycol was preferentially formed when the oxidation was performed at an alkaline pH. 5. The mechanism of the oxidation is discussed.
INTRODUCTION
A development of procedures for the selective degradation of specific bases in nucleic acids would evidently facilitate progress in nucleic acid research. For example, such procedures would be useful in studies on the correlation between chemical structures and biological functions of nucleic acids as well as in studies on their primary structures. Permanganate oxidation has been employed for the chemical degradation of deoxyribonucleic acids ~-4. In this reaction, when it is carried out under relatively vigorous conditions, pyrimidines and guanine residues in deoxyribonucleic acid are degraded, while adenine residues remain unaffected ~. Work by Jones and co-workers has shown that the permanganate oxidation of thymine under relatively vigorous conditions gives a mixture of various compounds s. Thus, cis-thymine glycol (cis-5,6-dihydroxy-5,6-dihydrothymine) was formed as a primary product of the oxidation, wheu the reaction was carried out at pH 7 or 9 and 37 ° for 19 h, and, in the course of the reaction, this glycol was further hydrolyzed and oxidized, yielding urea, acetol, pyruvaldehyde, pyruvic acid and formic acid. In our previous communication 4, we described a procedure of permanganate oxidation with which pyrimidine residues, especially thymine residues that are i * A p r e l i m i n a r y r e p o r t of a p a r t of this w o r k has been c o m m u n i c a t e d x.
Biochim. Biophys. Acta 213 (197 o) 1-13
2
S. III)A, H. H A Y A T S U
volved in the single-stranded region of a nucleic acid chain, can be selectively degraded. The procedure consisted of a mild oxidation with dilute potassium permanganate solution at neutral pH and o °. in order to elucidate the uature of this reaction and the properties <~f the reaction products, we have undertaken a detailed study of the permanganate oxidation of thymine. We have now found that oxidation of thymine with permanganate for a short period at pH 7 greatly reduces the COlnplexity of the reaction which has been experienced by previous workers. Only cis-thymine glycol and 5-hydroxy-5-methylbarbituric acid are the main products of the reaction. This is the first case in which the latter compound has been detected in the products of the permanganate oxidation of thymine. In this paper we describe the identification of these reaction products, properties of them, the pH dependence of the product distribution, and the mechanism of the reaction.
RIS,SU LTS
Prclimitzarv aJzah,sis o~ the lScrma~zgalzatc oxidatio~z o~ t%,mim: Fig. I shows that thymine was completely oxidized within 2 rain in o.o2 M KMnO 4 at pH 7 and o °*. in order to prevent any rise in the pH of the reaction mixture during the reaction, phosphate buffer had been added into the solution. Attempts to desalt the reaction solution by use of an ion-exchange resin failed. However, some of the organic solvents, especially acetone, were found to be effective in extracting the oxidation products. When the products were examined by means of
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F i g . I. The o x i d a t i o n of t h y m i n e w i t h p o t a s s i u n l p e r n l a n g a n a t e a t o ' a n d p H 7. T h y l n i n e ( o . o 1 4 3 M) w a s t r e a t e d w i t h p o t a s s i n m p e r m a n g a n a t e , a n d t h e d e c r e a s e in a b s o r b a n c e was d e t e r m i n e d a s p r e v i o u s l y d e s c r i b e d ~2. @, w i t h o.o2 M l i M n O ~ ; O , w i t h o . o I ~r K M n O , . F i g . 2. Cellulose c o l m n n c h r o m a t o g r a p h y of p e r m a n g a n a t e o x i d i z e d t h w n i n e . T h w n i n e ([ g) w a s oxidized with o.o2 M permanganate a s described in t h e t e x t and f r a c t i o n a t e ~ l . C o l u m n size: 4. ] c m ;,. 42 c m . g l u t i n g s o l v e n t : w a t e r - s a t d , n b u t a n o h 7 m l per f r a c t i o n w a s e l u t e d a t a f l o w r a t e of 23 f r a c t i o n s per clay. "~Vhen t r a c t i o n 167 h a d b e e n eluted, t h e s o l v e n t w a s s u b s t i t u t e d for b y a s o l v e n t n b u t a n o l - e t h a n o l w a t e r ( 4 : [ : 5 , b y v o h ) . H o w e v e r , no p e a k w a s c l u t e d t h e r e a f t e r . D e t e c t i o n of t h e p r o d u c t s in e a c h f r a c t i o n of t h e c o l u i n n c h r o m a t o g r a p h y w a s p e r f o r m e d b y m e a s u r i n g b o t h t h e u l t r a v i o l e t a b s o r p t i o n at 253 n m ( -) a n d t h e d r y w e i g h t ( ).
* In this c x p e r i n l e n t t h e o x i d a t i o n w a s t e r m i n a t e d b y a d d i t i o n of s o d i u m sulfite into tile r e a c t i o n m i x t u r e . S i n c e w e h a v e c u r r e n t l y d i s c o v e r e d t h a t sulfite r e a c t s w i t h uracil 6, w e t h o u g h t t h a t t h y m i n e , too, m i g h t r e a c t w i t h sulfite. U n d e r t h e c o n d i t i o n s e m p l o y e d in t h e p r e s e n t studies, h o w e \ er, t h y m i u e w a s found to be c o m p l e t e l y u n a f f e c t e d b y t h e p r e s e n c e of sulfite in t h e r e a c t i o n l n i x t l l r e .
lHochim. I3ioptlys. Acta, -'z 3 (I97O) [ - i 3
PERMANGANATE OXIDATION OF THYMINE
3
paper chromatography, three very weakly ultraviolet-absorbing components were detected which were designated Spots A, B and C. Neither thymine nor urea was detectable. It was noted that immediately after spraying alkali on the chromatogram both Spot A and Spot B, especially the former, became strongly ultravioletabsorbing. This phenomenon greatly facilitated the detection of these spots. It was observed that the yellow coloration of Spot A with the p-dimethylaminobenzaldehyde reagent, after prior spraying with alkali, was less pronounced than that of Spot B. The ureido compound, which was produced from Spot B after the alkali spray, gave a time-dependent color change on reaction with the p-dimethylaminobenzaldehyde reagent. This color change has been known to be characteristic of thymine glycoF. In addition to this, the RF values in several paper chromatographic systems 7-9 and the slow but distinct consumption of periodate S (an experiment performed on the chromatogram) have indicated that Spot B corresponded to cis-thymine glycol. Thus, in these preliminary experiments, Spot B was deduced to correspond to cis-thymine glycol, while the nature of Spot A and Spot C remained obscure.
Isolation and characterization o/the reaction products Next, the experiment was carried out on a larger scale and the products were isolated by use of cellulose column chromatography. As is shown in Fig. 2, four peaks were obtained. The first two peaks were discarded because of their small quantities in weight. The Peak- 3 fraction gave a pure crystalline compound which corresponded to Spot A. Properties of this material were consistent with those of 5-hydroxy5-methylbarbituric acid. An authentic specimen of 5-hydroxy-5-methylbarbituric acid 12was indistinguishable from this compound. Thus, a mixed m.p. was undepressed, then infrared spectra were superimposable, and their behavior in paper electrophoresis and paper chromatography was indistinguishable. The Peak- 4 compound, which corresponded to Spot B, was isolated pure in a crystalline form. It was identified as cis-thymine glycol in terms of its melting point 5, the mode of periodate consumptionS, s, infrared 10 and NMR spectra n, behavior in paper electrophoresis, and RF values in paper chromatography s-9. A study of mass spectrum of cis-thymine glycol also gave support for this structural assignment. In contrast to these successful isolations of the main reaction products, the isolation of the third product, corresponding to Spot C, was difficult to achieve because this compound stuck to the chromatographic column very strongly. However, from the following experimental facts this compound is believed to be methyltartronylurea. Thus, when 5-hydroxy-5-methylbarbituric acid was hydrolyzed with alkali, a compound, which exhibited the same RF value as that of Spot C in paper chromatography, was produced. That Spot C was derived exclusively from 5-hydroxy5-methylbarbituric acid and not from cis-thymine glycol was confirmed by the experiments performed with radioactive compounds (see below). In addition, RF values of Spot C are quite close to those values reported for methyltartronylurea 12..
Quantitative aspects of the reaction Further investigation of this oxidation was performed by use of E2-1*Cjthymine * P e r m a n g a n a t e o x i d a t i o n of t h y m i d i n e followed b y h y d r o l y s i s gives m e t h y l t a r t r o n y l u r e a d e o x y r i b o s i d e la. I n t h i s e x p e r i m e n t , t h e m e t h y l t a r t r o n y l u r e a d e o x y r i b o s i d e h a s b e e n isolated as a p o w d e r in a p u r e s t a t e a n d c h a r a c t e r i z e d well. T h i s fact c o n s t i t u t e s a c o m p l e m e n t to t h e a b o v e structural assignment.
Biochim. Biophys. Acta, 213 (197 o) 1-13
4
s. IIDA, H. HAYATSU
as the starting material. It was confirmed that the acetone extraction technique employed for the recovery of the products (Method A, see MATERIALSAND METHODS) did actually take up more than 95 % of the radioactivity into the acetone phase. From the results of the distribution of the radioactivity on the paper chromatogram, it can be concluded that, under the reaction conditions employed, thymine has been derived into only these three compounds described above.
The p H dependence o] the product distribution In the next study, the change of the product distribution as a function of the pH of the reaction mixture was investigated using the radioactive thymine. The results, presented in Fig. 3 and Table I, indicate that (I) 5-hydroxy-5-methylbarbituric acid
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Fig. 3. The permanganate oxidation of [2-14C~thymineat various pH values (Method B). [2-14C]thymine was treated with permanganate as described in MATI~RIALSAND METItODSunder Permanganate oxidation o[ [2-14C]thymine.Method B. A = 5-hydroxy-5-methylbarbituric acid; /3 = cisthymine glycol. is the predominant product when the oxidation is performed in an acidic solution, whereas cis-thymine glycol predominates in the alkaline reaction and (2) the formation of Spot C can be completely prevented under these conditions.
Properties o] 5-hydroxy-5-methylbarbituric acid and cis-thymine glycol In ultraviolet spectra, 5-hydroxy-5-methylbarbituric acid exhibited only an end absorption at neutral and acidic pH's (Fig. 4). In alkali (o.oi M NaOH), on the other hand, the spectrum showed a maximum in the neighbourhood of 243 nm. This is the reason why Spot A has strongly absorbed ultraviolet light at 253 nm after the alkali spray on the chromatogram. The 243-nm peak, however, diminished very rapidly, as is presented in Fig. 4. This was due to the hydrolysis of 5-hydroxy-5-methylbarbituric acid into methyltartronylurea. Fig. 5 presents the first-order rate decomposition of 5hydroxy-5-methylbarbituric acid at various pH values, as recorded by the loss of ab-
Bioehirn. Biophys. Aeta, 213 (197o) 1-13
PERMANGANATE OXIDATION OF THYMINE
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Fig. 4- Ultraviolet spectra of 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid a n d the t i m e - d e p e n d e n t change of its alkaline spectra. - - , s p e c t r u m at p H 8. 5, where hydrolysis of 5 - h y d r o x y - 5 - m e t h y l b a r bituric acid is v e r y slow; -- --, s p e c t r u m in w a t e r and in o. i M HC1 solution. 5 - H y d r o x y - 5 - m e t h y l b a r b i t u r i c acid solution was t r e a t e d w i t h a p p r o x , o.oi 1V[ N a O H , and the t i m e - d e p e n d e n t loss of a b s o r p t i o n at r o o m t e m p e r a t u r e was m e a s u r e d at a given w a v e l e n g t h (22o, 23o, 240, 25 o, 260, 270 and 280 nm). F r o m t h e d a t a collected in these studies, each spectral curve ( O , O , A) was determined. The time after the addition of alkali O , 3 ° sec; O , 2 rain; &, 5 min. Fig. 5. Stability of 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid at various pFI values. 5 - H y d r o x y - 5 - m e t h ylbarbituric acid w a s t r e a t e d at various p H values as described in MATERIALS AND METIIODS u n d e r Alkaline degradation o[ the oxidation products. A sample of i . lO -4 M solution was a d j u s t e d to the p H value indicated a n d the loss of a b s o r p t i o n at 243 n m was determined. O , p H 8.5; O , p H 9 . o ; A, p H IO.O;[], p H II.O; II, p H 12.2.
sorption at 243 nm. It is clear that the stronger the alkali, the more rapid is the decomposition. Therefore, the spectrum of the alkaline form of 5-hydroxy-5-methylbarbituric acid was determined at pH 8.5, where the hydrolysis was very slow (Fig. 4). This spectrum gave a maximum at 243 nm. The molecular extinction coefficient at 243 nm at pH II or 12 was determined by extrapolation (Fig. 5) and found to be 7.25. Io 3. The
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Fig. 6. Ultraviolet s p e c t r a of cis-thymine glycol. Solid line ( ) is t h e s p e c t r u m at p H 12.o a n d b r o k e n line (-- - - ) is t h e s p e c t r u m in w a t e r a n d in o.i 1V[ HC1 solution. Fig. 7. Stability of cis-thymine glycol at various p H values, cis-Thymine glycol was t r e a t e d at various p H values as described in MATERIALS AND METHODS u n d e r Alkaline degradation o/ the ~xidation products. A sample of i . io -4 M solution w a s a d j u s t e d to the p H value indicated and the loss of a b s o r p t i o n at 23o n m was determined. El, p H i i.o; m, p H i 1.9; O , p H 13.o; O , p H 14.o.
Biochim. Biophys. Acta, 213 (I97 o) 1-13
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OXIDATION PRODUCTS OF [2-14CJTHY'MINE
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The percentage of the oxidation p r o d u c t s was calculated f r o m t o t a l c o u n t s on t h e p a p e r c h r o m a t o g r a m , a n d is given in p a r e n t h e s e s .
D I S T R I B U T I O N OF T H E P E R M A N G A N A T E
TABLE I
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PERMANGANATE OXIDATION OF THYMINE
7
pKa value of this compound as determined by use of the pH-dependent change of the absorption at 243 nm was found to be approx. 7.5 (Fig. 8), whereas a value of 7.1 was obtained by means of the titration. Ultraviolet absorption spectra of cis-thymine glycol showed an end absorption in neutral and acidic solution, but in alkali it showed a maximum at 230 nm (Fig. 6). Fig. 7 presents the rate of hydrolytic decomposition of cis-thymine glycol at various pH values. Of note is that the loss of absorption at 230 nm in I M NaOH (designated pH 14.o in Fig. 7) does not follow the first-order rate, and, in particular, the decomposition is slower than that at pH 13. Although this peculiar phenomenon was repeatedly observed, no further investigation was made of this point. The pKa value of this compound was determined to be approx, lO.8 (Fig. 8).
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Fig. 8. The p/f~ values of 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid and c/s-thymine glycol. The molecular extinction coefficients of the oxidation p r o d u c t s at various p H values were obtained from Figs. 5 and 7. The extinction values at o t h e r p H values, which are n o t presented in Figs. 5 and 7, were also determined in a similar m a n n e r . These values were plotted and from the plots the pKa values of the oxidation p r o d u c t s were determined. S , 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid, at 243 n m (D, cis-thymine glycol, at 230 nm.
The stability of 5-hydroxy-5-methylbarbituric acid and cis-thymine glycol in neutral solution was compared. Incubation of the oxidized E2-14Clthymine at pH 7.1 and 4 °° followed by paper chromatographic analysis showed that 5-hydroxy-5-methylbarbituric acid gradually underwent hydrolysis giving methyltartronylurea, while cis-thymine glycol remained completely unaffected. In order to check the stability of cis-thymine glycol and of 5-hydroxy-5-methylbarbituric acid under the reaction conditions we employed for the oxidation of thymine, these compounds were separately treated with permanganate at both pH 4.3 and pH 7.0, for 2 rain at o °. It was found, when analyzed by paper chromatography, that both of these compounds had been practically unaffected by this treatment. This result indicates that once these two products were generated during the oxidation of thymine, they must have remained unchanged throughout the reaction.
DISCUSSION
The results presented in the preceding section have shown that the permanganate oxidation of thymine simply gives two products in a quantitative fashion if it is carried out under the conditions described above. This is in striking contrast to the complexity of the permanganate oxidation of thymine reported by JoNEs and co-workers, who characterized six compounds from the reaction mixture 5. These contrasting results might stem from differences in the reaction conditions. The length of the reaction period, 2 rain, employed in our experiment may not be considered to be Biochim. Biophys. Acta, 213 (197 o) 1-13
8
S. IIDA, H. HAYATSU
so strict, since both cis-thymine glycol and 5-hydroxy-5-methylbarbituric acid were found to be resistant to further 2-min treatment with fresh permanganate reagent. Regarding the 5-hydroxy-5-methylbarbituric acid, we have been able to determine its ultraviolet spectrum in weak alkali and to show that it possesses a maximum at 243 nm (Fig. 4). STUCKEY14 reported that he did not notice a maximum of this compound when he measured the spectrum in o.I M NaOH. Our finding that the 5hydroxy-5-methylbarbituric acid undergoes very rapid hydrolysis in alkaline solution clearly explains this apparent discrepancy between his observation and ours. The pH dependency of the product distribution (Fig. 3 and Table I) is noteworthy. It would be of interest to investigate whether such pH dependency can bc obtained with thymine residues that are the constituents of deoxyribonucleic acid. The stability of cis-thymine glycol in the permanganate treatment described above indicates that 5-hydroxy-5-methylbarbituric acid, of which the oxidation level is higher than that of cis-thymine glycol, has been formed not from cis-thymine glycol but directly from thymine. As could be expected, treatment of 5-hydroxy- 5methylbarbituric acid with potassium permanganate did not give cis-thymine glycol. It can therefore be concluded that each of these products of the permanganate oxidation of thymine is a primary product produced directly from thymine through independent pathways. o
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The stability of cis-thymine glycol in the aqueous solution under the conditions of the oxidation of thymine also shows that the cis-trans isomerization, which has been reported to occur slowly in an aqueous solution 7,s, did not take place under our reaction conditions*. * T r e a t m e n t of t h e cis-thymine glycol in an a q u e o u s s o l u t i o n for a long period, h o w e v e r , did yield a s m a l l a m o u n t of t h e trans-thymine glycol. This partial i s o m e r i z a t i o n t o o k place n o t o n l y in n e u t r a l s o l u t i o n b u t also in acid and in alkali solutions. These e x p e r i m e n t s are c o n s i s t e n t w i t h t h e p r e v i o u s r e p o r t t h a t t h e i s o m e r i z a t i o n of t h e cis-glycol i n t o the t r a n s i s o m e r occurs u p o n h e a t i n g of t h e f o r m e r at IOO° for 4 h in an a q u e o u s solutionL The characteristics of t h e transglycol o b t a i n e d b y us are also t h e s a m e as t h o s e reported p r e v i o u s l y . Thus, it gave a characteristic c o l o r a t i o n on a paper c h r o m a t o g r a m , w h e n it w a s s p r a y e d w i t h alkali and p - d i m e t h y l a m i n o b e n z a l d e h y d e reagent, successively. This glycol s h o w e d a n e g a t i v e reaction w i t h t h e periodate reagent. In a d d i t i o n to these k n o w n properties, trans-glycol w a s found to s h o w a ~max at 233 n m in alkaline media, w h i l e at n e u t r a l p H it e x h i b i t e d o n l y an end absorption. These spectral properties are a n a l o g o u s to t h o s e of cis-thymine glycol. W h e n t h e trans-thymine glycol w a s subjected to paper electrophoresis in borate buffer (pH 8.5), it s h o w e d no m o b i l i t y t o w a r d t h e anode, a prop e r t y c o n t r a s t i n g w i t h t h a t of cis isomer. These d a t a s u p p o r t t h e v i e w t h a t this isomeric t h y m i n e glycol is i n d e e d t h e trans-glycol.
Biochim. Biophys. Acta, 213 (197 o) 1-13
PERMANGANATE OXIDATION OF THYMINE
9
It should be pointed out that the permanganate oxidation of thymine bears a close similarity to the permanganate oxidation of olefins. With olefins, it has been known that both cis-diols and ketols are produced as the primary products of permanganate oxidation 15,16. With regard to the pH dependence of the product distribution, it has been reported that oleic acid gives its diol derivative in a quantitative fashion on treatment with permanganate in alkaline solution, while the main product at neutral pH is its ketol derivative 16. All these teatures quite resemble those of the permanganate oxidation of thymine. Therefore, the mechanism of the permanganate oxidation of thymine must be analogous to that proposed by WIBERG AND SAEGEBARTH17 for the permanganate oxidation of olefins. It involves an intermediate formation of a cyclic ester produced from the permanganate ion and the 5,6-double bond of thymine. While thymine is attacked by permanganate at its 5,6-double bond, pyrimidine nucleosides with exocyclic thio groups have been shown to be oxidized first at its thio group. For example, a brief permanganate treatment brings about the transformations of (I) 4-thiouridine into uridine-4-sulfonate ls,19 and (2) 2-thiouracil-I-arabinoside into 2,2'-cyclouridine (H. HAYATSU, unpublished results). It would be of interest to extend the present studies to the nucleoside and nucleotide levels and to examine the properties of the oxidized thymine nucleosides and nucleotides. Such studies would be valuable for the development of techniques which are useful as tools to investigate the function and the primary structure of nucleic acids. Experiments along these lines are now in progress. MATERIALS AND METHODS
Materials Thymine was purchased from Nutritional Biochemical Corp., Cleveland, Ohio. Reagent grade potassium permanganate, which was obtained from Wako Pure Chemical Industries, Osaka, Japan, was used throughout this work. E2-14ClThymine was purchased from New England Nuclear Corp., Boston, Mass. An authentic sample of 5-hydroxy-5-methylbarbituric acid was prepared according to DOUMAS AND BIGGS12. General methods Ascending chromatography was carried out on Toyo filter paper No. 53 (Toyo Roshi Co.). The following solvent systems were utilized: System A, n-butanol-water (86 : 14, v/v); B, n-propanol-water (IO :3, v/v)J C, n-butanol-ethanol-water (4 : I :5, by vol.); D, n-butanol-formic acid (7:3, v/v)-water (saturated, upper phase); E, ethylacetate-acetic acid-water (3:1:1, by vol.); F, diethyl ether-acetic acid-water (13 : 3 : I, by vol.) ; G, n-butanol-acetic acid-water (2 : I : I, by vol.) and H, n-butanolformic acid-water (lO:2:15, by vol.). Pyrimidine rings with saturated 5,6 C-C bonds were detected by a succesive spray of alkali, which opens the rings, and the p-dimethylaminobenzaldehyde reagent z°, giving coloration of the resulting ureido compounds. Urea and ureido compounds were detected by direct spraying with the benzaldehyde reagent, cis-Thymine glycol was located by means of a metaperiodate reagent ~1. Ultraviolet-absorbing compounds were located by scanning the chromatogram over an ultraviolet lamp which emits a 253-nm ray. Biochim. Biophys. Acta, 213 (197 o) 1-I3
IO
S. I I D A , H. H A Y A T S U
Spectrophotometric assay was carreid out using Cary Model I I and Beckman DU spectrophotometers, p H ' s were determined with a T6a D e m p a p H meter. Titration for the determination of p K , was performed in a Radiometer titrator. NMR spectra (60 MHz) were determined in hexadeuterodimethylsulfoxide, tetramethylsilane being used as an internal standard. Infrared analyses were performed on KBr pellets.
Rate o/reaction Oxidation was followed by the decrease in the ultraviolet absorption of the pyrimidine bases as previously described 4.
Permanganate oxidation o~ thymine: paper chromatographic studies Thymine (5o#moles) was dissolved in c.2 M phosphate buffer (2.5 ml) (pH 6.8), and 0.07 M potassium permanganate solution (I ml) was added to the solution. The reaction was allowed to proceed at o ° for 2 rain under ice cooling, and then it was stopped b y addition of I M sodium bisulfite. Manganese dioxide which precipitated was removed by centrifugation for IO min. A large excess of acetone was added to the supernatant solution to extract the products. After removal of inorganic salts by centrifugation, the acetone phase was taken up and evaporated to dryness. The residue was dissolved in water and examined b y means of paper chromatography. Three spots were observed which gave the following RF values in System A: Spot A, o.31; Spot B, o.21 and Spot C, 0.04. RF values of these products in other paper chromatographic systems are summarized in Table II.
TABLE
RF
II
VALUES
OF COMPOUNDS
Solvent system
RF value Thymine
Urea
5-Hydroxy-5methylbarbituric acid
cisThymine glycol
transThymine glycol
Methyltartronyl urea
A I3 C D E F O H
o.50 o.68 0.56 0.54 0.70 0.49 0.64 --
o.25 0.49 0.36 o.35 0.65 0.53 o.58 0.35
o.31 o.56 0.40 0.36 0.52 0.35 0.49 0.37
o.21 0.49 0.34 0.23 0.43
o.30 o.59 0.38 0.28 o.51 0.39 0.47
o.04* -o.I5" -0.65 0.52 -0.53
These
0.27 0.44 --
RF v a l u e s w e r e t h o s e o f t h e s o d i u m s a l t .
Under these reaction conditions, the final concentration of phosphate buffer used was o.143 M, ten times more concentrated than thymine, and the p H change during the reaction was less than 0.3. Independent experiments were performed to check the effect of the buffer concentration on the p H change during the reaction. Biochim. Biophys. Acta, 2 1 3 (197 o) 1 - 1 3
PERMANGANATE OXIDATION OF THYMINE
II
Isolation o/the oxidation products o] thymine Thymine (I g) was oxidized with potassium permanganate with vigorous shaking under ice cooling as described above. During the reaction, the pH of the reaction mixture was between 6.8 and 7.1, and its temperature was 15-17 °. Manganese dioxide which precipitated was removed by centrifugation for 15 min and the supernatant was concentrated under reduced pressure at below 300 . By extraction (twice) with a large excess of acetone, the reaction products were transferred into the acetone phase, most of the inorganic salts being removed as a semisolid. The acetone solution was evaporated under reduced pressure and was fractionated by means of cellulose column chromatography (see Fig. 2). In order to avoid the hydrolysis of the products, the chromatography was performed at approx. 4 °. The column was eluted with water-satd, n-butanol. The product in the fraction was detected by measurement of both its ultraviolet absorption at 253 nm and its dry weight after the fraction was evaporated to dryness. Four peaks were detected. The first (fractions 34-43) and the second (fractions 53-61) peaks weighed only 13 mg and 18 rag, respectively, and therefore were discarded. The third-peak compound (Spot A) (fractions 88-IO6), which was identified as 5-hydroxy-5-methylbarbituric acid as described below, was obtained in colorless crystals (recrystallized from ethanol); m.p. 226-227°; yield, 0.51 g (4° °/o). Found: C, 38.12; H, 3.72; N, 17.87. CsHeN204 requires: C. 37.98; H, 3.83; N, 17.72. Infrared spectrum: 339 o, 3250, 31oo and 3020 cm -J (v N - H and v O-H), 171o-175o cm -1 (broad, v C=O), 1179 and 1113 cm -1 (v C-O of 5-OH). NMR spectrum: 1.47 ppm (5-CH3), 6.07 ppm (5-OH) and lO.12 ppm (two protons, I- and 3-NH). Mass spectrum: A peak at m/e 158, corresponding to the molecular ion of 5-hydroxy-5-methylbarbituric acid, and several other strong peaks, namely, role 115, 87, 72, 44 and 43, were obtained. The peaks at role 115 and 87 corresponded to (M--CONH) + and to a fragment in which a carbonyl group was lost from the (M--CONH) + fragment, respectively. The fourth-peak compound (Spot B) (fractions I27-I57), cis-thymine glycol, was recrystallized from ethanol-water to colorless crystals; m.p. 215-216 ° (decomp.); yield, 0.45 g (35.5 %). Found: C, 37-75; H, 5.36; N, 17.52. CsHsN=O4 requires: C, 37.50; H, 5.04; N, 17.5o. Infrared spectrum; 34.36 and 3356 cm -1 (v O-H), 3236 cm -1 (v N-H), 1737, 17o 5 and 1671 cm -1 (v C=O), 117o and i i i i cm -1 (v C-O of 5-OH) and lO87 and lO53 cm -1 (v C-O of 6-OH). NMR spectrum: 1.25 ppm (5CHs), 4.36 ppm (6-H), 5.29 ppm (5-OH), 6.02 ppm (6-OH), 8.12 ppm (I-NH) and lO.O7 ppm (3-NH). Mass spectrum; although a peak corresponding to the molecular ion of thymine glycol was not obtained, a peak at m/e 161 which corresponded to ( M + I ) + was observed. Several strong peaks, namely, m/e 115, 89, 72, 46, 44 and 43, were also observed. The peak at m/e 89 of this glycol can be interpreted as corresponding to the peak at m/e 87 obtained from 5-hydroxy-5-methylbarbituric acid. Other peaks observed can be interpreted in accordance with the proposed structure.
Paper electrophoresis o/the reaction products Paper electrophoresis in 0.o2 M borate buffer (pH 8.5) for 2 h at 12 V/cm gave the following mobilities (in cm/h) toward the anode, cis-Thymine glycol, 2.3; transthymine glycol, o; 5-hydroxy-5-methylbarbituric acid, 2.8; ribose 1. 9 and urea, o. Biochim. Biophys. Acta, 213 (197 o) 1-13
12
s. IIDA, H. HAYATSU
In phosphate buffer, at pH 8.5 and 9.5 (1.5 h at 12 V/cm), cis-thymine glycol had little or no mobility toward the anode.
Alkaline degradation ol the oxidation products A 2" lO -4 M (or I. lO -3 M) solution of a reaction product was adjusted to an appropriate pH by addition of one volume of 0.02 M phosphate or phosphate-NaOH buffer. The time-dependent loss of absorption at room temperature (read against an appropriate blank) was measured and plotted on semi-log paper. From these plots, the value of the zero-time intercept was obtained by extrapolation, from which the molar extinction coefficient at the given wavelength was calculated.
Permanganate oxidation ol E2-14CJthymine Method A. A solution of ~2-14C~thymine was treated with permanganate as described above in the section Permanganate oxidation o/thymine: paper chromatographic studies. The final concentrations of the components were: I2-J4Clthymine, o.o143 M and 5 ° nC/o. 7 ml; sodium phosphate butter (pH 7) o.143 M; potassium permanganate, 0.02 M. The total volume was 0. 7 ml. After the work-up, the acetone phase was evaporated to dryness and the residue was dissolved in 0.5 ml of water. The recovery of the radioactivity at each step was as follows: the MnO 2 fraction, 4.7 %; the inorganic precipitate fraction, 0. 4 °/o and the acetone fraction, 98.4 ~o. Samples of the solution were examined by means of paper chromatography in System A. The paper was cut into pieces I cm wide and each counted in a Packard LiquidScintillation Counter. The distribution of the radioactivity on the chromatogram was found to be as follows: 5-hydroxy-5-methylbarbituric acid, 41.8 ~'o; cis-thymine glycol, 41.6 °/o and methyltartronylurea, 13. 9 ~o. Practically no radioactivity was located in areas other than these. Method B. In order to minimize the hydrolysis of the oxidized products, a modified method was developed. This method was employed in the studies of the pH dependence of the product distribution. Solutions of thymine in buffers of various pH values were treated with permanganate in the same manner as described above. The final concentrations of the components were identical with those of Method A. Total volume was 0. 7 ml. Buffers used were: Acetate buffer for pH 4.3 and pH 5.2 reactions; phosphate buffer for pH 6. 4 and pH 7.0 reactions and ammonium buffer for pH 8.6 reaction. Under these conditions, the pH change in each reaction mixture during the oxidation was found to be less than o.3. After the addition of 0. 5 M sodium bisulfite for the termination of the reaction, I M phosphate buffer (o.I nal), the pH of which was identical with that of the reaction solution, was added to the brown turbid mixture. Then approx. 5 ° m l of acetone were added to the mixture and the oxidized thymine was extracted into the acetone phase. The presence of phosphate ions effectively prevents the manganese ions from being extracted into the acetone phase. The radioactivity present in the starting material was quantitatively recovered in this acetone phase (Table I). This technique permits one to omit both the step of the removal of manganese dioxide by centrifugation and the subsequent step of the concentration of the aqueous solution. The acetone-extraction process was carried out under cooling in an ethanol-dry ice bath. After centrifugation of the acetone-added mixture for 8 min, the acetone phase was collected, evaporated to dryness, and the residue was dissolved in 0. 5 ml water and examined by paper chromatography. The total procedure described above can be completed within 30 rain.
Biochim. Biophys. Mcta, 213 (197o) i-13
PERMANGANATE OXIDATION OF "IHYMINE
13
Hydrolysis o[ the oxidized I2-14C~thymine in neutral solution A portion of the finally obtained residue, described in the preceding paragraph, was subjected to hydrolysis at neutral pH. Thus, it was incubated in o.I M phosphate buffer (pH 7.1) at 4 o°, aliquots were withdrawn at appropriate time intervals, and the products were extracted with acetone and examined by paper chromatography and by radioactivity counting. For example, in the case of E2-1*Clthymine which had been oxidized with permanganate at pH 7 and o °, the distribution of each product was as follows: 5-hydroxy-5-methylbarbituric acid, 58.6 %/0 time, 38. 9 %/ 20 rain, and 19.6 0/0/80 rain; thymine glycol, 39.9 %/0 time, 39.5 %/20 rain, and 41.1 0/0/80 rain and methyltartronylurea, 0.8 %/0 time, 19.6 0/0/20 rain, and 37.6 0/0/80 rain. Additional treatment o/ eis-thymine glycol and 5-hydroxy-5-methylbarbituric acid with potassium permanganate cis-Thymine glycol (25 #moles) or 5-hydroxy-5-methylbarbituric acid (25 #moles) was treated with permanganate. The reaction mixture was worked up in the same manner as described before in Permanganate oxidation o/ I2-14Clthymine. Method B. The resulting acetone phase was subjected to the paper chromatographic analysis. ACKNOWLEDGMENT
The authors are grateful to Professor T. Ukita for his encouragement throughout this work. REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21
H. HA.YATSU AND S. IIDA, Tetrahedron Letters, (1969) lO31. A. S. JONES AND C. R. BAYLEY, Trans. Faraday Sot., 55 (1959) 492. G. K. DARBY, A. S. JONES, J. R. TITTENSOR AND R. T. WALKER, Nature, 216 (1967) 793. H. HAYATSU AND T. UKITA, Biochem. Biophys. Res. Commun., 29 (1967) 556. ~V[.H. ]3ENN, B. CHATAMRA AND A. S. JONES, J. Chem. Soe., (196o) lOl 4. H. HAYATStl, Y. WATAYA AND K. KAI, J. Am. Chem. Soc., 92 (197 o) 724 . B. EKEET, Nature, 194 (1962) 278. D. BARSZCZ, Z. TRAMER AND I). SHUGKR, Acta Biochim. Polon., io (1963) 9. C. NOFRE AND A. CIER, Bull. Soe. Chim. France, (1966) 1326. C. NOFRE, ]Y[. MURAT AND A. CIER Bull. Soc. Chim. France, (1965) 1749. M. CHABRE, D. GAGNAIRE AND C. NOFRE, Bull. Soc. Chim. France, (1966) lO8. ]3. DOUMAS AND G. BIGGS, J. Biol. Chem., 237 (1962) 2306. S. IIDA AND H. I~IAYATSU, to be published. R. E. STUCKE¥, Quart. J. Pharm. Pharmacol., 15 (1942) 37 o. G. KING, J. Chem. Soc., (1936) 1788. J. E. COLEMAN, C. RICCIUTI AND D. SWERN, J. A~n. Chem. Soe., 78 (1956) 5342. K. B. WIBERG AND K. A. SAEGEBARTH, J. Am. Chem. Soc., 79 (1957) 2822. H. HAYA.TSU AND M. YANO, Tetrahedron Letters, (1969) 755. 1V[. YANO AND H. HAYATSU, Biochim. Biophys. Acta, 199 (197o) 303 • R. M. FINK, R. E. CLINE, C. MCGAUGHEY AND K. FINK, Anal. Chem., 28 (1956) 4. J. A. CIFONELLI AND F. SMITH, Anal. Chem., 26 (1954) 1132.
Biochim. Biophys. Acta, 213 (197 o) 1-13
BBA 96524
T H E PERMANGANATE OXIDATION OF THYMINE* S H I G E R U I I D A AND H I K O Y A H A Y A T S U
Faculty o/ Pharmaceutical Sciences, University oI Tokyo; Bunkyo-ku, Tokyo (Japan) (Received J a n u a r y i6th, 197 o)
SUMMARY
I. Mild permanganate oxidation of thymine yielded cis-thymine glycol (cis5,6-dihydroxy-5,6-dihydrothymine ) and 5-hydroxy-5-methylbarbituric acid. Under certain reaction conditions, only these two compounds were the products of the reaction. 2. While cis-thymine glycol was resistant to hydrolysis at neutral pH, 5-hydroxy-5-methylbarbituric acid readily underwent hydrolysis to give methyltartronylurea. 3. In this oxidation reaction, 5-hydroxy-5-methylbarbituric acid, which is higher in the oxidation level than thymine glycol, is formed not via the thymine glycol but directly from thymine. 4. When the oxidation of thymine was carried out at an acidic pH, 5-hydroxy5-methylbarbituric acid was predominantly produced, whereas thymine glycol was preferentially formed when the oxidation was performed at an alkaline pH. 5. The mechanism of the oxidation is discussed.
INTRODUCTION
A development of procedures for the selective degradation of specific bases in nucleic acids would evidently facilitate progress in nucleic acid research. For example, such procedures would be useful in studies on the correlation between chemical structures and biological functions of nucleic acids as well as in studies on their primary structures. Permanganate oxidation has been employed for the chemical degradation of deoxyribonucleic acids ~-4. In this reaction, when it is carried out under relatively vigorous conditions, pyrimidines and guanine residues in deoxyribonucleic acid are degraded, while adenine residues remain unaffected ~. Work by Jones and co-workers has shown that the permanganate oxidation of thymine under relatively vigorous conditions gives a mixture of various compounds s. Thus, cis-thymine glycol (cis-5,6-dihydroxy-5,6-dihydrothymine) was formed as a primary product of the oxidation, wheu the reaction was carried out at pH 7 or 9 and 37 ° for 19 h, and, in the course of the reaction, this glycol was further hydrolyzed and oxidized, yielding urea, acetol, pyruvaldehyde, pyruvic acid and formic acid. In our previous communication 4, we described a procedure of permanganate oxidation with which pyrimidine residues, especially thymine residues that are i * A p r e l i m i n a r y r e p o r t of a p a r t of this w o r k has been c o m m u n i c a t e d x.
Biochim. Biophys. Acta 213 (197 o) 1-13
2
S. III)A, H. H A Y A T S U
volved in the single-stranded region of a nucleic acid chain, can be selectively degraded. The procedure consisted of a mild oxidation with dilute potassium permanganate solution at neutral pH and o °. in order to elucidate the uature of this reaction and the properties <~f the reaction products, we have undertaken a detailed study of the permanganate oxidation of thymine. We have now found that oxidation of thymine with permanganate for a short period at pH 7 greatly reduces the COlnplexity of the reaction which has been experienced by previous workers. Only cis-thymine glycol and 5-hydroxy-5-methylbarbituric acid are the main products of the reaction. This is the first case in which the latter compound has been detected in the products of the permanganate oxidation of thymine. In this paper we describe the identification of these reaction products, properties of them, the pH dependence of the product distribution, and the mechanism of the reaction.
RIS,SU LTS
Prclimitzarv aJzah,sis o~ the lScrma~zgalzatc oxidatio~z o~ t%,mim: Fig. I shows that thymine was completely oxidized within 2 rain in o.o2 M KMnO 4 at pH 7 and o °*. in order to prevent any rise in the pH of the reaction mixture during the reaction, phosphate buffer had been added into the solution. Attempts to desalt the reaction solution by use of an ion-exchange resin failed. However, some of the organic solvents, especially acetone, were found to be effective in extracting the oxidation products. When the products were examined by means of
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Peak
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Time (rain)
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F i g . I. The o x i d a t i o n of t h y m i n e w i t h p o t a s s i u n l p e r n l a n g a n a t e a t o ' a n d p H 7. T h y l n i n e ( o . o 1 4 3 M) w a s t r e a t e d w i t h p o t a s s i n m p e r m a n g a n a t e , a n d t h e d e c r e a s e in a b s o r b a n c e was d e t e r m i n e d a s p r e v i o u s l y d e s c r i b e d ~2. @, w i t h o.o2 M l i M n O ~ ; O , w i t h o . o I ~r K M n O , . F i g . 2. Cellulose c o l m n n c h r o m a t o g r a p h y of p e r m a n g a n a t e o x i d i z e d t h w n i n e . T h w n i n e ([ g) w a s oxidized with o.o2 M permanganate a s described in t h e t e x t and f r a c t i o n a t e ~ l . C o l u m n size: 4. ] c m ;,. 42 c m . g l u t i n g s o l v e n t : w a t e r - s a t d , n b u t a n o h 7 m l per f r a c t i o n w a s e l u t e d a t a f l o w r a t e of 23 f r a c t i o n s per clay. "~Vhen t r a c t i o n 167 h a d b e e n eluted, t h e s o l v e n t w a s s u b s t i t u t e d for b y a s o l v e n t n b u t a n o l - e t h a n o l w a t e r ( 4 : [ : 5 , b y v o h ) . H o w e v e r , no p e a k w a s c l u t e d t h e r e a f t e r . D e t e c t i o n of t h e p r o d u c t s in e a c h f r a c t i o n of t h e c o l u i n n c h r o m a t o g r a p h y w a s p e r f o r m e d b y m e a s u r i n g b o t h t h e u l t r a v i o l e t a b s o r p t i o n at 253 n m ( -) a n d t h e d r y w e i g h t ( ).
* In this c x p e r i n l e n t t h e o x i d a t i o n w a s t e r m i n a t e d b y a d d i t i o n of s o d i u m sulfite into tile r e a c t i o n m i x t u r e . S i n c e w e h a v e c u r r e n t l y d i s c o v e r e d t h a t sulfite r e a c t s w i t h uracil 6, w e t h o u g h t t h a t t h y m i n e , too, m i g h t r e a c t w i t h sulfite. U n d e r t h e c o n d i t i o n s e m p l o y e d in t h e p r e s e n t studies, h o w e \ er, t h y m i u e w a s found to be c o m p l e t e l y u n a f f e c t e d b y t h e p r e s e n c e of sulfite in t h e r e a c t i o n l n i x t l l r e .
lHochim. I3ioptlys. Acta, -'z 3 (I97O) [ - i 3
PERMANGANATE OXIDATION OF THYMINE
3
paper chromatography, three very weakly ultraviolet-absorbing components were detected which were designated Spots A, B and C. Neither thymine nor urea was detectable. It was noted that immediately after spraying alkali on the chromatogram both Spot A and Spot B, especially the former, became strongly ultravioletabsorbing. This phenomenon greatly facilitated the detection of these spots. It was observed that the yellow coloration of Spot A with the p-dimethylaminobenzaldehyde reagent, after prior spraying with alkali, was less pronounced than that of Spot B. The ureido compound, which was produced from Spot B after the alkali spray, gave a time-dependent color change on reaction with the p-dimethylaminobenzaldehyde reagent. This color change has been known to be characteristic of thymine glycoF. In addition to this, the RF values in several paper chromatographic systems 7-9 and the slow but distinct consumption of periodate S (an experiment performed on the chromatogram) have indicated that Spot B corresponded to cis-thymine glycol. Thus, in these preliminary experiments, Spot B was deduced to correspond to cis-thymine glycol, while the nature of Spot A and Spot C remained obscure.
Isolation and characterization o/the reaction products Next, the experiment was carried out on a larger scale and the products were isolated by use of cellulose column chromatography. As is shown in Fig. 2, four peaks were obtained. The first two peaks were discarded because of their small quantities in weight. The Peak- 3 fraction gave a pure crystalline compound which corresponded to Spot A. Properties of this material were consistent with those of 5-hydroxy5-methylbarbituric acid. An authentic specimen of 5-hydroxy-5-methylbarbituric acid 12was indistinguishable from this compound. Thus, a mixed m.p. was undepressed, then infrared spectra were superimposable, and their behavior in paper electrophoresis and paper chromatography was indistinguishable. The Peak- 4 compound, which corresponded to Spot B, was isolated pure in a crystalline form. It was identified as cis-thymine glycol in terms of its melting point 5, the mode of periodate consumptionS, s, infrared 10 and NMR spectra n, behavior in paper electrophoresis, and RF values in paper chromatography s-9. A study of mass spectrum of cis-thymine glycol also gave support for this structural assignment. In contrast to these successful isolations of the main reaction products, the isolation of the third product, corresponding to Spot C, was difficult to achieve because this compound stuck to the chromatographic column very strongly. However, from the following experimental facts this compound is believed to be methyltartronylurea. Thus, when 5-hydroxy-5-methylbarbituric acid was hydrolyzed with alkali, a compound, which exhibited the same RF value as that of Spot C in paper chromatography, was produced. That Spot C was derived exclusively from 5-hydroxy5-methylbarbituric acid and not from cis-thymine glycol was confirmed by the experiments performed with radioactive compounds (see below). In addition, RF values of Spot C are quite close to those values reported for methyltartronylurea 12..
Quantitative aspects of the reaction Further investigation of this oxidation was performed by use of E2-1*Cjthymine * P e r m a n g a n a t e o x i d a t i o n of t h y m i d i n e followed b y h y d r o l y s i s gives m e t h y l t a r t r o n y l u r e a d e o x y r i b o s i d e la. I n t h i s e x p e r i m e n t , t h e m e t h y l t a r t r o n y l u r e a d e o x y r i b o s i d e h a s b e e n isolated as a p o w d e r in a p u r e s t a t e a n d c h a r a c t e r i z e d well. T h i s fact c o n s t i t u t e s a c o m p l e m e n t to t h e a b o v e structural assignment.
Biochim. Biophys. Acta, 213 (197 o) 1-13
4
s. IIDA, H. HAYATSU
as the starting material. It was confirmed that the acetone extraction technique employed for the recovery of the products (Method A, see MATERIALSAND METHODS) did actually take up more than 95 % of the radioactivity into the acetone phase. From the results of the distribution of the radioactivity on the paper chromatogram, it can be concluded that, under the reaction conditions employed, thymine has been derived into only these three compounds described above.
The p H dependence o] the product distribution In the next study, the change of the product distribution as a function of the pH of the reaction mixture was investigated using the radioactive thymine. The results, presented in Fig. 3 and Table I, indicate that (I) 5-hydroxy-5-methylbarbituric acid
'2
g° 4/
7.o
"~ 0/~ . . . . . . . .
2[° If
n n n n ~ a n
pH
®®
8.0 I
Fig. 3. The permanganate oxidation of [2-14C~thymineat various pH values (Method B). [2-14C]thymine was treated with permanganate as described in MATI~RIALSAND METItODSunder Permanganate oxidation o[ [2-14C]thymine.Method B. A = 5-hydroxy-5-methylbarbituric acid; /3 = cisthymine glycol. is the predominant product when the oxidation is performed in an acidic solution, whereas cis-thymine glycol predominates in the alkaline reaction and (2) the formation of Spot C can be completely prevented under these conditions.
Properties o] 5-hydroxy-5-methylbarbituric acid and cis-thymine glycol In ultraviolet spectra, 5-hydroxy-5-methylbarbituric acid exhibited only an end absorption at neutral and acidic pH's (Fig. 4). In alkali (o.oi M NaOH), on the other hand, the spectrum showed a maximum in the neighbourhood of 243 nm. This is the reason why Spot A has strongly absorbed ultraviolet light at 253 nm after the alkali spray on the chromatogram. The 243-nm peak, however, diminished very rapidly, as is presented in Fig. 4. This was due to the hydrolysis of 5-hydroxy-5-methylbarbituric acid into methyltartronylurea. Fig. 5 presents the first-order rate decomposition of 5hydroxy-5-methylbarbituric acid at various pH values, as recorded by the loss of ab-
Bioehirn. Biophys. Aeta, 213 (197o) 1-13
PERMANGANATE OXIDATION OF THYMINE
1.£
0.5
0.5
E
O
210
~
23O
-.-
...,_
_ -.t_..-.:_~_~_--z-.~-~
25O '~(nm)
270
0
5
Time (rain)
10
Fig. 4- Ultraviolet spectra of 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid a n d the t i m e - d e p e n d e n t change of its alkaline spectra. - - , s p e c t r u m at p H 8. 5, where hydrolysis of 5 - h y d r o x y - 5 - m e t h y l b a r bituric acid is v e r y slow; -- --, s p e c t r u m in w a t e r and in o. i M HC1 solution. 5 - H y d r o x y - 5 - m e t h y l b a r b i t u r i c acid solution was t r e a t e d w i t h a p p r o x , o.oi 1V[ N a O H , and the t i m e - d e p e n d e n t loss of a b s o r p t i o n at r o o m t e m p e r a t u r e was m e a s u r e d at a given w a v e l e n g t h (22o, 23o, 240, 25 o, 260, 270 and 280 nm). F r o m t h e d a t a collected in these studies, each spectral curve ( O , O , A) was determined. The time after the addition of alkali O , 3 ° sec; O , 2 rain; &, 5 min. Fig. 5. Stability of 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid at various pFI values. 5 - H y d r o x y - 5 - m e t h ylbarbituric acid w a s t r e a t e d at various p H values as described in MATERIALS AND METIIODS u n d e r Alkaline degradation o[ the oxidation products. A sample of i . lO -4 M solution was a d j u s t e d to the p H value indicated a n d the loss of a b s o r p t i o n at 243 n m was determined. O , p H 8.5; O , p H 9 . o ; A, p H IO.O;[], p H II.O; II, p H 12.2.
sorption at 243 nm. It is clear that the stronger the alkali, the more rapid is the decomposition. Therefore, the spectrum of the alkaline form of 5-hydroxy-5-methylbarbituric acid was determined at pH 8.5, where the hydrolysis was very slow (Fig. 4). This spectrum gave a maximum at 243 nm. The molecular extinction coefficient at 243 nm at pH II or 12 was determined by extrapolation (Fig. 5) and found to be 7.25. Io 3. The
tE
1,C
\ 0.5
0.5
\
\ 0
21'0
g 230
7~( n m )
25O
270 Time (min)
Fig. 6. Ultraviolet s p e c t r a of cis-thymine glycol. Solid line ( ) is t h e s p e c t r u m at p H 12.o a n d b r o k e n line (-- - - ) is t h e s p e c t r u m in w a t e r a n d in o.i 1V[ HC1 solution. Fig. 7. Stability of cis-thymine glycol at various p H values, cis-Thymine glycol was t r e a t e d at various p H values as described in MATERIALS AND METHODS u n d e r Alkaline degradation o/ the ~xidation products. A sample of i . io -4 M solution w a s a d j u s t e d to the p H value indicated and the loss of a b s o r p t i o n at 23o n m was determined. El, p H i i.o; m, p H i 1.9; O , p H 13.o; O , p H 14.o.
Biochim. Biophys. Acta, 213 (I97 o) 1-13
~o
©
t~ L~
OXIDATION PRODUCTS OF [2-14CJTHY'MINE
i5o (0.2)
(58-1)
390
(0.4)
pH7.o
pH8.6
57 5 ° 0
850 (0.9)
pH6. 4
i2 3o0 (13.2)
61 500 (67.7)
68 3oo (73-4)
181o (1.9)
pH5.z
83 800 (86.2)
5-Hydroxy-5methylbarbituric acid
251o (2.6)
Thymine
78 ooo (83.7)
(40.0)
39 600
27 500 (30.3)
19 7oo (21.2)
8 21o (8.4)
cis-Thymine glycol
Radioactivity (countMmin) [ound for
pH4-3
Condition
20o0 (2.2)
(1.1)
113o
800 (0.9)
2290 (2.5)
46o (0.5)
~Xlethyltartronyl urea
63 ° (0.7)
(0.4)
420
18o (o.2)
98o (1.1)
(2.3)
219o
The rest
93 ioo
99 ooo
90 800
93 IOO
97 20o
Total counts on the chromatogram
3600
54 °
94 °
94o
200
Counts in the acetone-insoluble /faction
The percentage of the oxidation p r o d u c t s was calculated f r o m t o t a l c o u n t s on t h e p a p e r c h r o m a t o g r a m , a n d is given in p a r e n t h e s e s .
D I S T R I B U T I O N OF T H E P E R M A N G A N A T E
TABLE I
~q Gq
.>
o~
O",
PERMANGANATE OXIDATION OF THYMINE
7
pKa value of this compound as determined by use of the pH-dependent change of the absorption at 243 nm was found to be approx. 7.5 (Fig. 8), whereas a value of 7.1 was obtained by means of the titration. Ultraviolet absorption spectra of cis-thymine glycol showed an end absorption in neutral and acidic solution, but in alkali it showed a maximum at 230 nm (Fig. 6). Fig. 7 presents the rate of hydrolytic decomposition of cis-thymine glycol at various pH values. Of note is that the loss of absorption at 230 nm in I M NaOH (designated pH 14.o in Fig. 7) does not follow the first-order rate, and, in particular, the decomposition is slower than that at pH 13. Although this peculiar phenomenon was repeatedly observed, no further investigation was made of this point. The pKa value of this compound was determined to be approx, lO.8 (Fig. 8).
O.51'0 f
pfl a ,,,.,
7.~
-" ~
o
r.~
, ,'~'~6T" ? 8,'-~,10. . . . .12 pH
0 4
14
Fig. 8. The p/f~ values of 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid and c/s-thymine glycol. The molecular extinction coefficients of the oxidation p r o d u c t s at various p H values were obtained from Figs. 5 and 7. The extinction values at o t h e r p H values, which are n o t presented in Figs. 5 and 7, were also determined in a similar m a n n e r . These values were plotted and from the plots the pKa values of the oxidation p r o d u c t s were determined. S , 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid, at 243 n m (D, cis-thymine glycol, at 230 nm.
The stability of 5-hydroxy-5-methylbarbituric acid and cis-thymine glycol in neutral solution was compared. Incubation of the oxidized E2-14Clthymine at pH 7.1 and 4 °° followed by paper chromatographic analysis showed that 5-hydroxy-5-methylbarbituric acid gradually underwent hydrolysis giving methyltartronylurea, while cis-thymine glycol remained completely unaffected. In order to check the stability of cis-thymine glycol and of 5-hydroxy-5-methylbarbituric acid under the reaction conditions we employed for the oxidation of thymine, these compounds were separately treated with permanganate at both pH 4.3 and pH 7.0, for 2 rain at o °. It was found, when analyzed by paper chromatography, that both of these compounds had been practically unaffected by this treatment. This result indicates that once these two products were generated during the oxidation of thymine, they must have remained unchanged throughout the reaction.
DISCUSSION
The results presented in the preceding section have shown that the permanganate oxidation of thymine simply gives two products in a quantitative fashion if it is carried out under the conditions described above. This is in striking contrast to the complexity of the permanganate oxidation of thymine reported by JoNEs and co-workers, who characterized six compounds from the reaction mixture 5. These contrasting results might stem from differences in the reaction conditions. The length of the reaction period, 2 rain, employed in our experiment may not be considered to be Biochim. Biophys. Acta, 213 (197 o) 1-13
8
S. IIDA, H. HAYATSU
so strict, since both cis-thymine glycol and 5-hydroxy-5-methylbarbituric acid were found to be resistant to further 2-min treatment with fresh permanganate reagent. Regarding the 5-hydroxy-5-methylbarbituric acid, we have been able to determine its ultraviolet spectrum in weak alkali and to show that it possesses a maximum at 243 nm (Fig. 4). STUCKEY14 reported that he did not notice a maximum of this compound when he measured the spectrum in o.I M NaOH. Our finding that the 5hydroxy-5-methylbarbituric acid undergoes very rapid hydrolysis in alkaline solution clearly explains this apparent discrepancy between his observation and ours. The pH dependency of the product distribution (Fig. 3 and Table I) is noteworthy. It would be of interest to investigate whether such pH dependency can bc obtained with thymine residues that are the constituents of deoxyribonucleic acid. The stability of cis-thymine glycol in the permanganate treatment described above indicates that 5-hydroxy-5-methylbarbituric acid, of which the oxidation level is higher than that of cis-thymine glycol, has been formed not from cis-thymine glycol but directly from thymine. As could be expected, treatment of 5-hydroxy- 5methylbarbituric acid with potassium permanganate did not give cis-thymine glycol. It can therefore be concluded that each of these products of the permanganate oxidation of thymine is a primary product produced directly from thymine through independent pathways. o
./~/c HN ~ O H HN " / I ~ c H 3 H I
H
MnO4
H3
ll" o
~...,,j/C H3
o'>'-- ""<'[~OH H~N o H .E
9~ >
H2N-C-NH'C-CO1' oil OH'COOH "IV"
S c h e m e i. I, t h y m i n e ; II, cis t h y m i n e glycol; IfI, 5 - h y d r o x y - 5 - m e t h y l b a r b i t u r i c acid; IV, m e t h yltartronylurea.
The stability of cis-thymine glycol in the aqueous solution under the conditions of the oxidation of thymine also shows that the cis-trans isomerization, which has been reported to occur slowly in an aqueous solution 7,s, did not take place under our reaction conditions*. * T r e a t m e n t of t h e cis-thymine glycol in an a q u e o u s s o l u t i o n for a long period, h o w e v e r , did yield a s m a l l a m o u n t of t h e trans-thymine glycol. This partial i s o m e r i z a t i o n t o o k place n o t o n l y in n e u t r a l s o l u t i o n b u t also in acid and in alkali solutions. These e x p e r i m e n t s are c o n s i s t e n t w i t h t h e p r e v i o u s r e p o r t t h a t t h e i s o m e r i z a t i o n of t h e cis-glycol i n t o the t r a n s i s o m e r occurs u p o n h e a t i n g of t h e f o r m e r at IOO° for 4 h in an a q u e o u s solutionL The characteristics of t h e transglycol o b t a i n e d b y us are also t h e s a m e as t h o s e reported p r e v i o u s l y . Thus, it gave a characteristic c o l o r a t i o n on a paper c h r o m a t o g r a m , w h e n it w a s s p r a y e d w i t h alkali and p - d i m e t h y l a m i n o b e n z a l d e h y d e reagent, successively. This glycol s h o w e d a n e g a t i v e reaction w i t h t h e periodate reagent. In a d d i t i o n to these k n o w n properties, trans-glycol w a s found to s h o w a ~max at 233 n m in alkaline media, w h i l e at n e u t r a l p H it e x h i b i t e d o n l y an end absorption. These spectral properties are a n a l o g o u s to t h o s e of cis-thymine glycol. W h e n t h e trans-thymine glycol w a s subjected to paper electrophoresis in borate buffer (pH 8.5), it s h o w e d no m o b i l i t y t o w a r d t h e anode, a prop e r t y c o n t r a s t i n g w i t h t h a t of cis isomer. These d a t a s u p p o r t t h e v i e w t h a t this isomeric t h y m i n e glycol is i n d e e d t h e trans-glycol.
Biochim. Biophys. Acta, 213 (197 o) 1-13
PERMANGANATE OXIDATION OF THYMINE
9
It should be pointed out that the permanganate oxidation of thymine bears a close similarity to the permanganate oxidation of olefins. With olefins, it has been known that both cis-diols and ketols are produced as the primary products of permanganate oxidation 15,16. With regard to the pH dependence of the product distribution, it has been reported that oleic acid gives its diol derivative in a quantitative fashion on treatment with permanganate in alkaline solution, while the main product at neutral pH is its ketol derivative 16. All these teatures quite resemble those of the permanganate oxidation of thymine. Therefore, the mechanism of the permanganate oxidation of thymine must be analogous to that proposed by WIBERG AND SAEGEBARTH17 for the permanganate oxidation of olefins. It involves an intermediate formation of a cyclic ester produced from the permanganate ion and the 5,6-double bond of thymine. While thymine is attacked by permanganate at its 5,6-double bond, pyrimidine nucleosides with exocyclic thio groups have been shown to be oxidized first at its thio group. For example, a brief permanganate treatment brings about the transformations of (I) 4-thiouridine into uridine-4-sulfonate ls,19 and (2) 2-thiouracil-I-arabinoside into 2,2'-cyclouridine (H. HAYATSU, unpublished results). It would be of interest to extend the present studies to the nucleoside and nucleotide levels and to examine the properties of the oxidized thymine nucleosides and nucleotides. Such studies would be valuable for the development of techniques which are useful as tools to investigate the function and the primary structure of nucleic acids. Experiments along these lines are now in progress. MATERIALS AND METHODS
Materials Thymine was purchased from Nutritional Biochemical Corp., Cleveland, Ohio. Reagent grade potassium permanganate, which was obtained from Wako Pure Chemical Industries, Osaka, Japan, was used throughout this work. E2-14ClThymine was purchased from New England Nuclear Corp., Boston, Mass. An authentic sample of 5-hydroxy-5-methylbarbituric acid was prepared according to DOUMAS AND BIGGS12. General methods Ascending chromatography was carried out on Toyo filter paper No. 53 (Toyo Roshi Co.). The following solvent systems were utilized: System A, n-butanol-water (86 : 14, v/v); B, n-propanol-water (IO :3, v/v)J C, n-butanol-ethanol-water (4 : I :5, by vol.); D, n-butanol-formic acid (7:3, v/v)-water (saturated, upper phase); E, ethylacetate-acetic acid-water (3:1:1, by vol.); F, diethyl ether-acetic acid-water (13 : 3 : I, by vol.) ; G, n-butanol-acetic acid-water (2 : I : I, by vol.) and H, n-butanolformic acid-water (lO:2:15, by vol.). Pyrimidine rings with saturated 5,6 C-C bonds were detected by a succesive spray of alkali, which opens the rings, and the p-dimethylaminobenzaldehyde reagent z°, giving coloration of the resulting ureido compounds. Urea and ureido compounds were detected by direct spraying with the benzaldehyde reagent, cis-Thymine glycol was located by means of a metaperiodate reagent ~1. Ultraviolet-absorbing compounds were located by scanning the chromatogram over an ultraviolet lamp which emits a 253-nm ray. Biochim. Biophys. Acta, 213 (197 o) 1-I3
IO
S. I I D A , H. H A Y A T S U
Spectrophotometric assay was carreid out using Cary Model I I and Beckman DU spectrophotometers, p H ' s were determined with a T6a D e m p a p H meter. Titration for the determination of p K , was performed in a Radiometer titrator. NMR spectra (60 MHz) were determined in hexadeuterodimethylsulfoxide, tetramethylsilane being used as an internal standard. Infrared analyses were performed on KBr pellets.
Rate o/reaction Oxidation was followed by the decrease in the ultraviolet absorption of the pyrimidine bases as previously described 4.
Permanganate oxidation o~ thymine: paper chromatographic studies Thymine (5o#moles) was dissolved in c.2 M phosphate buffer (2.5 ml) (pH 6.8), and 0.07 M potassium permanganate solution (I ml) was added to the solution. The reaction was allowed to proceed at o ° for 2 rain under ice cooling, and then it was stopped b y addition of I M sodium bisulfite. Manganese dioxide which precipitated was removed by centrifugation for IO min. A large excess of acetone was added to the supernatant solution to extract the products. After removal of inorganic salts by centrifugation, the acetone phase was taken up and evaporated to dryness. The residue was dissolved in water and examined b y means of paper chromatography. Three spots were observed which gave the following RF values in System A: Spot A, o.31; Spot B, o.21 and Spot C, 0.04. RF values of these products in other paper chromatographic systems are summarized in Table II.
TABLE
RF
II
VALUES
OF COMPOUNDS
Solvent system
RF value Thymine
Urea
5-Hydroxy-5methylbarbituric acid
cisThymine glycol
transThymine glycol
Methyltartronyl urea
A I3 C D E F O H
o.50 o.68 0.56 0.54 0.70 0.49 0.64 --
o.25 0.49 0.36 o.35 0.65 0.53 o.58 0.35
o.31 o.56 0.40 0.36 0.52 0.35 0.49 0.37
o.21 0.49 0.34 0.23 0.43
o.30 o.59 0.38 0.28 o.51 0.39 0.47
o.04* -o.I5" -0.65 0.52 -0.53
These
0.27 0.44 --
RF v a l u e s w e r e t h o s e o f t h e s o d i u m s a l t .
Under these reaction conditions, the final concentration of phosphate buffer used was o.143 M, ten times more concentrated than thymine, and the p H change during the reaction was less than 0.3. Independent experiments were performed to check the effect of the buffer concentration on the p H change during the reaction. Biochim. Biophys. Acta, 2 1 3 (197 o) 1 - 1 3
PERMANGANATE OXIDATION OF THYMINE
II
Isolation o/the oxidation products o] thymine Thymine (I g) was oxidized with potassium permanganate with vigorous shaking under ice cooling as described above. During the reaction, the pH of the reaction mixture was between 6.8 and 7.1, and its temperature was 15-17 °. Manganese dioxide which precipitated was removed by centrifugation for 15 min and the supernatant was concentrated under reduced pressure at below 300 . By extraction (twice) with a large excess of acetone, the reaction products were transferred into the acetone phase, most of the inorganic salts being removed as a semisolid. The acetone solution was evaporated under reduced pressure and was fractionated by means of cellulose column chromatography (see Fig. 2). In order to avoid the hydrolysis of the products, the chromatography was performed at approx. 4 °. The column was eluted with water-satd, n-butanol. The product in the fraction was detected by measurement of both its ultraviolet absorption at 253 nm and its dry weight after the fraction was evaporated to dryness. Four peaks were detected. The first (fractions 34-43) and the second (fractions 53-61) peaks weighed only 13 mg and 18 rag, respectively, and therefore were discarded. The third-peak compound (Spot A) (fractions 88-IO6), which was identified as 5-hydroxy-5-methylbarbituric acid as described below, was obtained in colorless crystals (recrystallized from ethanol); m.p. 226-227°; yield, 0.51 g (4° °/o). Found: C, 38.12; H, 3.72; N, 17.87. CsHeN204 requires: C. 37.98; H, 3.83; N, 17.72. Infrared spectrum: 339 o, 3250, 31oo and 3020 cm -J (v N - H and v O-H), 171o-175o cm -1 (broad, v C=O), 1179 and 1113 cm -1 (v C-O of 5-OH). NMR spectrum: 1.47 ppm (5-CH3), 6.07 ppm (5-OH) and lO.12 ppm (two protons, I- and 3-NH). Mass spectrum: A peak at m/e 158, corresponding to the molecular ion of 5-hydroxy-5-methylbarbituric acid, and several other strong peaks, namely, role 115, 87, 72, 44 and 43, were obtained. The peaks at role 115 and 87 corresponded to (M--CONH) + and to a fragment in which a carbonyl group was lost from the (M--CONH) + fragment, respectively. The fourth-peak compound (Spot B) (fractions I27-I57), cis-thymine glycol, was recrystallized from ethanol-water to colorless crystals; m.p. 215-216 ° (decomp.); yield, 0.45 g (35.5 %). Found: C, 37-75; H, 5.36; N, 17.52. CsHsN=O4 requires: C, 37.50; H, 5.04; N, 17.5o. Infrared spectrum; 34.36 and 3356 cm -1 (v O-H), 3236 cm -1 (v N-H), 1737, 17o 5 and 1671 cm -1 (v C=O), 117o and i i i i cm -1 (v C-O of 5-OH) and lO87 and lO53 cm -1 (v C-O of 6-OH). NMR spectrum: 1.25 ppm (5CHs), 4.36 ppm (6-H), 5.29 ppm (5-OH), 6.02 ppm (6-OH), 8.12 ppm (I-NH) and lO.O7 ppm (3-NH). Mass spectrum; although a peak corresponding to the molecular ion of thymine glycol was not obtained, a peak at m/e 161 which corresponded to ( M + I ) + was observed. Several strong peaks, namely, m/e 115, 89, 72, 46, 44 and 43, were also observed. The peak at m/e 89 of this glycol can be interpreted as corresponding to the peak at m/e 87 obtained from 5-hydroxy-5-methylbarbituric acid. Other peaks observed can be interpreted in accordance with the proposed structure.
Paper electrophoresis o/the reaction products Paper electrophoresis in 0.o2 M borate buffer (pH 8.5) for 2 h at 12 V/cm gave the following mobilities (in cm/h) toward the anode, cis-Thymine glycol, 2.3; transthymine glycol, o; 5-hydroxy-5-methylbarbituric acid, 2.8; ribose 1. 9 and urea, o. Biochim. Biophys. Acta, 213 (197 o) 1-13
12
s. IIDA, H. HAYATSU
In phosphate buffer, at pH 8.5 and 9.5 (1.5 h at 12 V/cm), cis-thymine glycol had little or no mobility toward the anode.
Alkaline degradation ol the oxidation products A 2" lO -4 M (or I. lO -3 M) solution of a reaction product was adjusted to an appropriate pH by addition of one volume of 0.02 M phosphate or phosphate-NaOH buffer. The time-dependent loss of absorption at room temperature (read against an appropriate blank) was measured and plotted on semi-log paper. From these plots, the value of the zero-time intercept was obtained by extrapolation, from which the molar extinction coefficient at the given wavelength was calculated.
Permanganate oxidation ol E2-14CJthymine Method A. A solution of ~2-14C~thymine was treated with permanganate as described above in the section Permanganate oxidation o/thymine: paper chromatographic studies. The final concentrations of the components were: I2-J4Clthymine, o.o143 M and 5 ° nC/o. 7 ml; sodium phosphate butter (pH 7) o.143 M; potassium permanganate, 0.02 M. The total volume was 0. 7 ml. After the work-up, the acetone phase was evaporated to dryness and the residue was dissolved in 0.5 ml of water. The recovery of the radioactivity at each step was as follows: the MnO 2 fraction, 4.7 %; the inorganic precipitate fraction, 0. 4 °/o and the acetone fraction, 98.4 ~o. Samples of the solution were examined by means of paper chromatography in System A. The paper was cut into pieces I cm wide and each counted in a Packard LiquidScintillation Counter. The distribution of the radioactivity on the chromatogram was found to be as follows: 5-hydroxy-5-methylbarbituric acid, 41.8 ~'o; cis-thymine glycol, 41.6 °/o and methyltartronylurea, 13. 9 ~o. Practically no radioactivity was located in areas other than these. Method B. In order to minimize the hydrolysis of the oxidized products, a modified method was developed. This method was employed in the studies of the pH dependence of the product distribution. Solutions of thymine in buffers of various pH values were treated with permanganate in the same manner as described above. The final concentrations of the components were identical with those of Method A. Total volume was 0. 7 ml. Buffers used were: Acetate buffer for pH 4.3 and pH 5.2 reactions; phosphate buffer for pH 6. 4 and pH 7.0 reactions and ammonium buffer for pH 8.6 reaction. Under these conditions, the pH change in each reaction mixture during the oxidation was found to be less than o.3. After the addition of 0. 5 M sodium bisulfite for the termination of the reaction, I M phosphate buffer (o.I nal), the pH of which was identical with that of the reaction solution, was added to the brown turbid mixture. Then approx. 5 ° m l of acetone were added to the mixture and the oxidized thymine was extracted into the acetone phase. The presence of phosphate ions effectively prevents the manganese ions from being extracted into the acetone phase. The radioactivity present in the starting material was quantitatively recovered in this acetone phase (Table I). This technique permits one to omit both the step of the removal of manganese dioxide by centrifugation and the subsequent step of the concentration of the aqueous solution. The acetone-extraction process was carried out under cooling in an ethanol-dry ice bath. After centrifugation of the acetone-added mixture for 8 min, the acetone phase was collected, evaporated to dryness, and the residue was dissolved in 0. 5 ml water and examined by paper chromatography. The total procedure described above can be completed within 30 rain.
Biochim. Biophys. Mcta, 213 (197o) i-13
PERMANGANATE OXIDATION OF "IHYMINE
13
Hydrolysis o[ the oxidized I2-14C~thymine in neutral solution A portion of the finally obtained residue, described in the preceding paragraph, was subjected to hydrolysis at neutral pH. Thus, it was incubated in o.I M phosphate buffer (pH 7.1) at 4 o°, aliquots were withdrawn at appropriate time intervals, and the products were extracted with acetone and examined by paper chromatography and by radioactivity counting. For example, in the case of E2-1*Clthymine which had been oxidized with permanganate at pH 7 and o °, the distribution of each product was as follows: 5-hydroxy-5-methylbarbituric acid, 58.6 %/0 time, 38. 9 %/ 20 rain, and 19.6 0/0/80 rain; thymine glycol, 39.9 %/0 time, 39.5 %/20 rain, and 41.1 0/0/80 rain and methyltartronylurea, 0.8 %/0 time, 19.6 0/0/20 rain, and 37.6 0/0/80 rain. Additional treatment o/ eis-thymine glycol and 5-hydroxy-5-methylbarbituric acid with potassium permanganate cis-Thymine glycol (25 #moles) or 5-hydroxy-5-methylbarbituric acid (25 #moles) was treated with permanganate. The reaction mixture was worked up in the same manner as described before in Permanganate oxidation o/ I2-14Clthymine. Method B. The resulting acetone phase was subjected to the paper chromatographic analysis. ACKNOWLEDGMENT
The authors are grateful to Professor T. Ukita for his encouragement throughout this work. REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21
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