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Photochemical transformation of 6-chlorouracil and some alkylated analogues
BIOCFIIMICA ET BIOPHY'SICA ACTA
I57
BBA 97073
PHOTOCHEMICAL TRANSFORMATION OF 6-CHLOROURACIL AND SOME A L K Y L A T E D ANALOGUES Z. K A Z I M I E R C Z U K AND D. SHUG-AR
Department o/ Biophysics, Institute o/ Experimental Physics, University o/ Warsaw, and Institute o/Biochemistry and Biophysics, Academy of Sciences, Warsaw (Poland) (Received July 6th, I971)
SUMMARY.
The monoanionic, but not the neutral, form of 6-chlorouracil in aqueous medium undergoes quantitative photochemical transformation to barbituric acid. Similarly, I-methyl-6-chlorouracil and 3-methyl-6-chlorouracil monoanions are transformed to I (3)-methylbarbituric acid, and 5-ethyl-6-chlorouracil to 5-ethylbarbituric acid, all in quantitative yield. The quantum yields for these reactions are quite high, 0.05 to 0.09, and unaltered over the wavelength range 254-280 nm, or b y a Io-fold change of concentration of the initial substances. The rates of photochemical transformation are independent of the O H - concentration, but exhibit a marked deuterium isotope effect. A reaction scheme consistent with the foregoing, and other, data is the photohydration of the 5,6 bond of the excited singlet state of the chlorouracil monoanion, followed b y the very rapid dark elimination of C1- to give barbitufic acid. The findings are discussed in relation to the photochemistry of 5-halogeno analogues of thymine.
INTRODUCTIO N
The photochemical transformations of 5-halogenouracils have been extensively investigated, largely because of the fact that 5-bromouracil and 5-iodouracil are base analogues of thymine and undergo incorporation into cellular or viral DNA, whereas 5-fluorouracil is a base analogue of uracil and can replace the latter in RNA. The replacement of thymine or uracil in a nucleic acid b y a 5-halogenouracil is usually accompanied by a pronounced modification in radiation sensitivity, as estimated by either biological or physicochemical criteria (ref. i, and references cited therein). However, the nature of the photoproducts of the 5-halogenouracils, and the reaction mechanisms involved, have not been satisfactorily clarified. In the case of 5-bromo- and 5-iodouracils, dehalogenation is observed, with partial formation of uracilZ,3; at least in the case of 5-iodouracil the generation of a free radical at the C-5 of the pyrimidine ring is involved ~. Dehalogenation is likewise observed with 5fluorouracil; the reaction in this instance has been shown to be wavelength dependent 4-~. At wavelengths to the red of 270 nm, a unique photoproduct is formed which has been identified as the hydrate, 5-fluoro-5,6-dihydro-6-hydroxyuracil; and this product can be further photochemically transformed b y irradiation at shorter wavelengths to eliminate fluoride anion with simultaneous formation of barbituric acid. Biochim. Biophys. Acta, 254 (197 I) 157-166
I5~
z. KAZIMIERCZUK,D. SHU(;AR
During the course of a study on the specific N-methylation of 6-chlorouracil, the products of which are intermediates in the synthesis of purines and pteridines 7, it was noted that ultraviolet irradiation of aqueous solutions of 6-chlorouracil and its alkylated derivatives led to conversion to new products with relatively high quantum yields. This finding was rather unexpected since, under analogous conditions, 5-chlorouracil is remarkably radiation resistant, being transformed (to unidentified products) with a quantum yield of less than IO-4 (M. FIKUS AND D. S~IUGAR, unpublished observations).
MATERIALSAND METHODS 6-Chlorouracil was prepared according to the method of CRESSWELL AND WOODs, and 5-ethyl-6-chlorouracil as described elsewhere 9. The preparation of 3methyl-6-chlorouracil, the previously unknown I-methyl-6-chlorouracil, and 1,3dimethyl-6-chlorouracil was based on the selective methylation of 6-chlorouraciU. The properties of the samples of 5-ethylbarbituric acid and I(3)-methylbarbituric acid, used as reference compounds and as filters, are described elsewhere 9,1°. All compounds were checked b y melting points and chromatography in several solvent systems. Ascending paper chromatography was employed with W h a t m a n paper No. I and following solvent systems: (A), water-saturated n-butanol; (B) water-saturated n-butanol with ammonia in the gas phase; (C) n-butanol-water-acetic acid (7 : 2 : I,
v/v/v). Phosphate buffers were employed in the alkaline pH range, where maintenance of constant pH was essential. A Radiometer PHM22 compensating pH meter with semi-micro glass electrode was used for controlling pH. Solutions were irradiated in Io-mm or I-mm path length quartz spectrophotometer cuvettes, the latter being employed when irradiated solutions were to be chromatographed, or for following rates at a lo-fold higher concentration, h~radiation involved the u.~e of three types of sources, as follows: (a) A Hanau Q7oo medium pressure mercury lamp with filters as indicated to isolate a given spectral region. (b) A Thermal Syndicate 254 nm mercury resonance lamp, with a sodium acetate filter to eliminate wavelengths below 230 nm. The intensity of this lamp at the cuvette surface was 7.5 " IOle quanta/cm2 per rain at 254 nm. (c) A Jarrell-Ash f I :3-5 grating monochromatgr with a I5o-W Hg-xenon lamp provided with a specially constructed condensing reflector for illumination of the entrance slit. The exit slit was set at 2 ram, corresponding to a band-width of 6 nm. This sorce was used to irradiate solutions in semi-micro cuvettes of width 4 mm, the solution being stirred continuously with a fine wire. Under these conditions the effective average intensity over the solution surface was 3.2 • lO15 quanta/cm 2 per min at 28o nm. Light intensities for sources (b) and (c) were estimated to about 5 % with the aid of a neutral aqueous solution of IO-* ~ uridine, using a quantum yield for uridine hydration of o.o21, known to be independent of the wavelength n. Actinometry was carried out in the same cuvettes used for irradiating the solutions under study. Biochim. Biophys. Acta, 254 (1971) 157-I66
PHOTOCHEMISTRY OF 6-CHLOROURACILS
159
RESULTS
Irradiation of 6-chlorouracil, lO -4 M in aqueous medium, pH approx. 6, at 254 nm led to the gradual disappearance of the characteristic absorption maximum at 283 nm, with the concomitant appearance of a new band centred at about 258 nm. Following irradiation, acidification of the solution to pH 2 with HC1 liquidated the 258 nm band which, however, was restored on neutralization of the solution. This behaviour of the 258 nm band is reminiscent of that for barbituric acid 1°. Paper chromatography of the irradiated solution, using Whatman No. I with various solvent systems (Table 2) demonstrated the presence of starting material and a second spot with an R~. identical with that for an authentic sample of barbituric acid. TABLE
I
ASCENDING RESPONDING
PAPER
CHROMATOGRAPHY
OF SOME DERIVATIVES
BARB1TUEIC ACIDS PHOTOCHEMICALLY
Compound
6-Chlorouracil B a r b i t u r i e acid 3-Methyl-6-chlorouracil i-Methyl-6-chlorouracil i ( 3 ) - M e t h y l b a r b i t u r i c acid 5-Ethyl-6-chl°r°uracil 5 - E t h y l b a r b i t u r i c acid
DERIVED
OF 6 - C H L O R O U R A C I L
AND
THE
COR-
FROM THEM
R F in solvent system A
B
C
o.54 o.12 o.77 o.62 0.49 o.84 0.44
o.63 o.21 o.76 -0.48 o.76 o.36
o.78 o.5o o.83 o.57 0.52 o.83 0.65
Since the pKa of 6-chlorouracilis 5.7 (ref. 12), a solution at pH 6 necessarily includes a mixture of the neutral and monoanionic forms. Irradiation of a solution of 6-chlorouracil at pH values below 6 led to a marked decrease in the rate of photochemical transformation and, at pH values below 4, no barbituric acid could be detected amongst the photoproducts. B y contrast, irradiation in buffered medium at pH values above 7, where 6chlorouracil is virtually in the form of the monoanion, led to an increase in the rate of photolysis, and in the extent of formation of barbituric acid. However, under these conditions, barbituric acid itself is exclusively in the form of the monoanion (pK 3.9, see ref. Io), with an absorption maximum at 258 nm and emax of 21.5" lOs, so that ez54nm is 18 • 103. Consequently the barbituric acid formed during irradiation of 6-chlorouracil is itself subject to photochemical transformation, when irradiated at 254 nm. Separate trials demonstrated that barbituric acid monoanion is considerably more radiation resistant than 6-chlorouracil, with a quantum yield for transformation at least an order of magnitude lower (c/. ref. Ii). But it appeared simpler to eliminate the possible complicating effects of irradiation on the barbituric acid formed, by irradiating at wavelengthts to the red of the long wavelength shoulder of barbituric acid monoanion, i.e. to the red of 275 nm. A lO -4 M solution of 6-chlorouracil in 0.02 M buffer, pH 8.7, was first irradiated with the Hanau Q7oo lamp through a Schott-Jena WG-7 filter cutting off completely at 265 nm. Subsequently this filter was replaced b y a 2-ram layer of 5 " IO-~ IV[ barBiochim. Biophys. Acta, 254 (197 I) I 5 7 - I 6 6
160
Z. KAZIMIERCZUK, D. SHUGAR
bituric acid monoanion (pH about 7)- The optical density of this filter is so high, about 2o A25s nm units, that it effectively cuts out radiation which would normally be adsorbed by barbituric acid formed in the irradiated solution, while the change in transmission during the irradiation times employed is negligible. Under these conditions, the course of photochemical transformation of 6-chlorouracil with time proceeded as depicted in Fig. I. It will be noted, from the two isosbestic points formed at 267.5 and 232 rim, that a single ultraviolet-absorbing photoproduct is formed from 6-chlorouraeil. Paper chromatography did, in fact, confirm the formation of only one ultiaviolet-absorbiug photoproduct (Table I), with RF values corresponding to authentic barbituric acid, further confirmed b y spectral examination of the eluates. That barbituric acid was the only product formed was supported b y a comparison of the optical density at ~.rr~x of the initial solution of 6-chlorouracil to that of the presumed barbituric acid spectrum following complete photolysis of the solution. From Fig. I, the absorbance of the initial 6-chlorouracil solution is o.82, while that of the photoproduct formed after 35 min irradiation is 1.36. The ratio of the latter to the former is 1.66. The emax of 6-chlorouracil monoanion is 12.5" IOa, while A I.l,
1.3
1.2
1.2
1.1
1.1
1.2 0.9
~9 0.8
t(in)
o.t
(11 ~s
s
0~ o3
l0
d (14
~____~0
~3
Q~ k\ o.1
20 s
220 230 2/,0 "250 260 270 280 290 300 3%0 3')0
AInrn)
O~ 220
2O
~o 2~o 2~o 2~o 2~o 280 ~o 300 glo A(nm}
Fig. I. Course of conversion w i t h t i m e of irradiation, w i t h source (a), of IO-1 or lO -8 M 6-chlorouracil m o n o a n i o n in o.o2 M p h o s p h a t e buffer (pH 8.7) to b a r b i t u r i c acid m o n o a n i o n . The radiation w a s filtered t h r o u g h a 2 - m m layer of 5 " I ° - S M b a r b i t u r i c acid monoanion, so t h a t b a r b i t u r i c acid f o r m e d in the reaction e u v e t t e a b s o r b e d no radiation. Figures denote irradiation times in minutes. N o t e isosbestic p o i n t s a t 267. 5 n m a n d 232 nm, p o i n t i n g to q u a n t i t a t i v e f o r m a t i o n of b a r b i t u r i c acid f r o m 6-chlorouracil. Following completion of reaction, solution w a s acidified to pFI 2 to give a b s o r p t i o n s p e c t r u m of n e u t r a l f o r m of b a r b i t u r i c acid (dashed line). Fig. 2. Course of t r a n s f o r m a t i o n of lO -41V[ 3-methyl-6-chlorouracil m o n o a n i o n in o.o2 M phosp h a t e buffer (pH 8.7), to I ( 3 ) - m e t h y l b a r b i t u r i c acid b y irradiation w i t h source (a), Q-7oo merc u r y lamp, t h r o u g h a 2 - m m layer of 5 " lO-8 IV[ I ( 3 ) - m e t h y l b a r b i t u r i c acid m o n o a n i o n . F i g u r e s denote irradiation t i m e s in rain. Isosbestic p o i n t s at 268 n m and 232 n m point to f o r m a t i o n of only one u l t r a v i o l e t - a b s o r b i n g p h o t o p r o d u c t , I ( 3 ) - m e t h y l b a r b i t u r i c acid.
Biochim. Biophys. Acta, 254 (1971) 157-166
PHOTOCHEMISTRY OF 6-CHLOROURACILS
161
that for barbituric acid monoanion is 21.5 • IOa (ref. IO), the resulting ratio being 1.72. The agreement is seen to be excellent. Analogous results were obtained on irradiation, under similar conditions, of 3-methyl-6-chlorouracil (Fig. 2) and 5-ethyl-6-chlorouracil (Fig. 3), both at appropriate pH values to give the monoanionic forms. The former was irradiated through a (I)3-methyl barbituric acid monoanion filter, which provides reasonably good protection of the I (3)-methylbarbituric acid expected as photoproduct. The latter was irradiated through a filter of 5-ethylbarbituric acid to protect the 5-ethylbarbituric acid expected from photolysis of 5-ethyl-6-chlorouracil. From Fig. 2 it will be observed that the absorbance of the initial 3-methyl-6chlorouracil is 0.70, while that of the product formed is 1.o7, giving a ratio of 1.53. The extinction coefficients at 2max of 3-methyl-6-chlorouracil monoanion and 3 (I)methylbarbituric acid are 12.4 and 20. 3 (refs. 12 and IO), respectively, so that their ratio is 1.64, in reasonable agreement with the observed value of 1.53, and again testifying to quantitative transformation of 3-methyl-6-chlorouracil to 3(I)-methyl barbituric acid. From Fig. 3, the observed ratio of absorbances at the maxima of the initial 5-ethyl-6-chlorouracil and the photoproduct formed following its disappearance is 1.71. This is to be compared with the ratio of the extinction coefficients at 2max of A
A
t.1
1.0
O.9
Q~
0.6
0.7
Qsj (1.5
~ ~4
25 75
o~-
\.\
~ / / ,
,
\\\
0.2
i
220 ~30 240 2gO 260 270 280 "290 300 310 a(nrnl
"220 "230 2~0 2S0 260 270 280 290 300 3t0 3"20 ~(.m)
Fig. 3. Course of t r a n s f o r m a t i o n of lO -41Vf 5-ethyl-6-chlorouracil m o n o a n i o n in 0.02 ]Vf p h o s p h a t e buffer (pH 8.7) to 5-ethylbarbituric acid m o n o a n i o n b y irradiation w i t h source (a) t h r o u g h a 2 - m m layer of 5 " lO-S ]Vf 5 - e t h y l b a r b i t u r i c acid monoanion. Figures denote irradiation times in min. Isosbestic p o i n t s at 279 n m and 235 n m p o i n t to f o r m a t i o n of only one ultravioleta b s o r b i n g product, 5 - e t h y l b a r b i t u r i c acid. Fig. 4. Course of conversion of lO -41Vf I-methyl-6-chlorouracil m o n o a n i o n in o.oi ]Y[ N a O H (pH a b o u t x2) to I ( 3 ) - m e t h y l b a r b i t u r i c acid b y irradiation w i t h source (a) t h r o u g h a 2-ram layer of 5 " 10 -3 M of i - m e t h y l b a r b i t u r i c acid monoanion. Figures denote irradiation time in min. Note isosbestic p o i n t s at 268 n m and 238 nm, p o i n t i n g to f o r m a t i o n of only one u l t r a v i o l e t - a b s o r b i n g p h o t o p r o d u c t , i - m e t h y l b a r b i t u r i c acid. Following 75 rain irradiation, b r e a k a w a y from t h e isosb e s t i c p o i n t occurred due to p h o t o l y s i s of p h o t o p r o d u c t . Hence at this p o i n t the solution was acidified to p H 2 to liquidate t h e a b s o r p t i o n s p e c t r u m of the I - m e t h y l b a r b i t u r i c acid p h o t o p r o duct, a n d t h u s reveal the s p e c t r u m of the unreacted I-methyl-6-chlorouracil (neutral form), w h i c h was e s t i m a t e d q u a n t i t a t i v e l y from its a b s o r b a n c e and k n o w n extinction coefficient.
Biochim. Biophys. Acta, 254 (1971) 157-166
162
z. KAZIMIERCZUK, D. SHUGAR
5-ethyl-6-ehlorouraeil (11. 9 • lO s, see ref. 9) and 5-ethylbarbituric acid (21 • io ~, see ref. 9), which is 1.77. For I-methyl-6-chlorouracil the same photochemical transformation was found, but in this instance the observations were somewhat complicated b y the fact that the absorption spectrum of the monoanion closely overlaps that of the product, I-methylbarbituric acid monoanion (Fig. 4)- Irradiation of I-methyl-6-ehlorouracil monoanion at p H 12 (o.oi M NaOH, pK of starting product 9.05) through a I-methylbarbituric acid monoanion filter proceeded with quantitative conversion to I-methylbarbituric acid only to the point where about 60 0/o conversion had occurred. Following this, a breakaway from the isosbestic point at 268 nm demonstrated the simultaneous photolysis of the i-methylbarbituric acid monoanion product in the reaction mixture. Consequently, following photolysis to the point where d e p a r t m e from the isosbestic point occurred, the solution was acidified to p H 2 to liquidate the spectrum of I-methylbarbituric acid monoanion, and to simultaneously place in evidence the spectrum at this p H of the neutral form of the remaining I-methyl-6-ehlorouracil (see Fig. 4). Subsequent calculation from the spectra of the amount of I-metbyl-6chlorouracil which had disappeared after 60 °/o photolysis, and the amount of barbituric acid formed, using their known extinction coefficients, demonstrated a difference between the two of only 7 9/o• For all four 6-chlorouracil monoanion derivatives, the rate and course of photochemical transformation was unaltered when irradiation was conducted at concentrations of lO -4 M (Io-mm cuvette) and lO -3 M (I-ram cuvette), the cuvettes being suitably positioned with regard to the source so that the incident intensities were the same. The reaction rates and products were also unchanged in the presence of air or nitrogen, the latter of which was flushed through the solutions prior to, and during irradiation. The liberation of CI-, which would be anticipated from the conversion of ehlorouracil to barbituric acid, was verified qualitatively during the course of the reactions b y periodic additions of AgNOs to precipitate AgC1. No attempt was made to quantitatively estimate C1-formation in view of the quantitative character" of the spectral data and confirmation of these b y chromatography. Liberation of C1- as the anion was further confirmed b y the observation that irradiation in unbuffered medium led to acidification during the course of the reaction; when the initial pH was uot for removed from the pK, the rate of photochemical transformation progressively decreased during irradiation. By contrast, when the reactions were conducted in buffered medium, at buffer concentrations sufficient to counteract the C1- liberated, reaction rates were found to be relatively unaffected b y an increase in the p H of the medium for p H values well above ( > I p H unit) the p H of the irradiated derivative, i.e. the reaction rate was not dependent on the O H - concentration.
Quantum yields With the aid of sources (b) and (c), measurements of quantum yields were carried out at 254 and 280 nm b y following quantitatively b y spectral methods the disappearance of starting product and appearance of the appropriate barbituric acid photoproduct, for low degrees of photolysis. The measured values of q~ are listed in Biochim, Biophys. Acta, 254 (1971) 157-166
163
PHOTOCHEMISTRY OF 6-CHLOROURACILS
Table II. For I-methyl-6-ehlorouracil, the intensity of the 28o-nm source was too low to permit of a n y reasonable estimation of qs. However, it is of interest that for the other three 6-chlorouracil derivatives, the quantum yields are essentially similar at the two wavelengths. This is further supported b y an examination of Figs. I, 2 and 3, where irradiation made use of a broad band of wavelengths from the filtered source (a). It will be noted that the relative rates of photolysis of 6-chlorouracil, 3-methyl-6chlorouracil and 5-ethyl-6-chlorouracil follow the same order as the quantum yields given in Table II.
TABLE II QUANTUM
YIELDS
RESPONDING
FOR
TRANSFORMATION
BARBITURIC
Compound
6-Chlorouracil i-Methyl-6-chlorouracil 3-Methy 1-6-chlorouracil I, 3-Dimethyl-6-chlorouracil 5-Ethyl-6-chlorouracil a b e a
OF 6-CHLOROURACIL
MONOANION
DERIVATIVES
TO C O R -
ACID MONOANIONS IN AQUEOUS MEDIUM
pK
5.75 9.05 5.95 -6.5
p H o[ irradiated solution
¢s 254 nm
280 nm
8.7b 12.o 8.7b 6. 5
0.060 0.060 o.o44 o.oi 0.090
0.075 -0.055 -0.o9o
8.7°
Product
Barbituric acid I (3)-Methylbarbituric acid i (3)-Methylbarburic acid __e
5 - E t h y l b a r b i t u r i c acid
Values to a b o u t 4 - i o %. Q u a n t u m yield essentially unaltered over p H range 7.8-i 1.3. Unidentified, b u t n o t 1,3-dimethylbarbituric acid. No change in q u a n t u m yield over p H range 7.8-12.
Kinetics and deuterium isotope e/Ject The photochemical conversion, or dechlorination, of the various 6-chlorouracils to the corresponding barbituric acids, since it is independent of the O H - concentration, probably involves a reaction with water and would therefore be expected to exhibit pseudo first-order kinetics. This is supported b y the fact that the reaction rates are unchanged over a Io-fold range in concentrations. Unfortunately it proved difficult to examine directly the reaction order, since the 254-nm source was in the wavelength range where absorption spectra of starting material and product overlap; while at 280 nm the intensity of the monochromator at our disposal was such t h a t inordinately long exposure times would have been required. If, however, the reaction does involve water, an isotope effect might be anticipated. Fig. 5 exhibits the course of disappearance of 6-chlorouracil with source (c) and a barbituric acid filter, when the reaction was run at p H 8.7 in H20 and p2H 8.7 in 2H20. It will be noted t h a t the rate of disappearance of 6-chlorouracil is appreciably slower in 2H20; if w e t a k e the slopes of these curves at zero time, then KH,o/ K2H,O = 2.1. When the reactions were run with source Co), 254 rim, the corresponding isotope effects for 6-chlorouracil, 3-methyl-6-chlorouracil and 5-ethyl-6-chlorouracil were 1.8, 1.85, and 1.8, respectively. Biochim. Biophys. Acta, 254 (1971) 157-166
164
Z. KAZIMIERCZUK, I). 5;H1T(;.~1{
,4
0.5
~4
H
0.3
2
0
~
0.~
0.1
1
2
3
4
5
6
7 8 t (rain)
9
10 1t
12 13
Fig. 5- D e u t e r i u m isotope effect for p h o t o c h e m i c a l conversion of 6-chlorouracil m o n o a n i o n to b a r b i t u r i e acid m o n o a n i o n as m e a s u r e d b y course of disappearance of i • lO -4 M 6-chlorouracil in HzO (at p H 8.7) and in 2H~O (at p i H 8.7), on irradiation w i t h source (a) t h r o u g h a filter consisting of a 2 - m m layer of 5 " lO-8 M b a r b i t u r i c acid monoanion. R a t e of disappearance m e a s u r e d b y decrease in a b s o r b a n c e at Jtmax. I r r a d i a t i o n w i t h source (b), 254 rim, gave similar results for relative rates in H~O and 2H20.
DISCUSSION
Before considering the possible mechanisms for the photochemical dechlorination of the various 6-chlorouracils, it should be noted that the ground state reactive monoanionic forms are not identical for all of them, but are divided into two groups, as in Scheme I(see refs. 7, 12). O
O
~ N ~ R 1
:
H R I = H~ R2=CH 3
O
CI
O
N
I
CI
Or" 1~1= CH2CH3,R2~-H or RI:
R2= 1-1
CH 3
Scheme I
If we recall (Table I I ) that, for each compound, the rate of photochemical transformation is essentially unaffected b y an increase in p H (in the p H range in which the reactive monoanionie forms exist), this implies that the photoreaction rate is independent of the O H - concentration. It is of interest in this connection that all the chlorouracil analogues exhibit remarkable stability in the ground state, even in hot acid and alkali 13. A plausible mechanism for the photochemical dechlorination reaction m a y therefore be presented as in Scheme 2 for the case of 6-chlorouracil, and involving hydration of the 5,6 bond, followed b y elimination of HC]. We have, of course, no direct evidence for the presumed hydrated intermediate, shown in brackets. Biochim. Biophys..4cta, 254 (1971) 157-166
PHOTOCHEMISTRY OF 6-CHLOROURACILS
165
o
H N( + I-t2o ©
ci
---
{
H
OH Cl
~
/H
___
-HCt O"
"N"
"O
I
H
Scheme 2
The absence of an oxygen effect m a y be taken to imply that the presumed hydration proceeds via the excited singlet state of the 6-chlorouracil monoanion, in accordance with the photohydration of uracil and cytosine, which are generally accepted as involving the excited singlets. It remains to establish whether the elimination of the C1- monoanion is photochemically induced, as in the case of hydrated 5-fluorouracil, which is stable at neutral p H but photochemically eliminates F - on irradiation at 254 nm to also give barbituric acid a. The data in Table I I provide an apparently unequivocal answer to this question. The presumed hydrated intermediate in Scheme 2 should, like hydrated 5-fluorouracil 4 or 2,4-diketo-5,6-dihydropyrimidines in general 14, exhibit low or negligible ultraviolet absorption at 280 nm relative to 254 rim. Notwithstanding this, the q u a n t u m yields at 280 nm, for those three compounds for which measurements were possible, are not inferior to those at 254 nm (Table II). It follows that the elimination of C1- from the presumed hydrated intermediate must be a dark reaction which is extremely rapid, so that the values given in Table I I must represent quant u m yields for the hydration reaction. This is indirectly supported b y the observed deuterium isotope effect for the overall reaction in 2H20 , b y analogy with the corresponding deuterium isotope effects foi the photohydration of the 5,6 bonds of uracil n and 5-fluorouracil analogues 4. It is of supplementary interest that, in contrast to the 6-chlorouracil derivatives embraced in this investigation, 5-fluorouracil will readily undergo hydration either in the neutral or the monoanionic form 5. The extreme instability of the presumed hydrated intermediate shown in Scheme 2 is in agreement with the fact that 5,6-dihydroderivatives of 6-halogenouracils are unknown, whereas those of 5-halogenouracils are readily prepared. An a t t e m p t was actually made to obtain the 5,6-dihydro derivatives of 6-chlorouracil b y photochemical reduction with K B H 4 as described b y CERUTTI et al. 15 for photochemical reduction of uracil ring. The results were completely negative, the only consequence being the reaction of K B H 4 with the barbituric acid photoproduct. Finally, we should like to emphasize that, apart from the interest of the foregoing reactions in relation to the photochemical behaviour of 5-halogenouracils, the photochemical dechlorination reactions herein described are quite unusual in themselves; and, from a perusal of the literature, are conspicuous by their absence. ACKNOWLEDGMENTS
We are indebted to Mgr. T. Kulikowski for the samples of 5-ethyl-6-chlorouracil and 5-ethylbarbituric acid; to Dr. Ewa Sztumpf-Kulikowskafor discussions; and to Mgr. K. Berens for assistance with the source reflector for the monochromator. This investigation was supported in part b y the World Health Organization, The Wellcome Trust, and the Agricultural Research Service, U.S. Department of Agriculture. Biochim. Biophys. Acta, 254 (1971) 157-166
166
z. KAZIMIERCZUK, D. SHUGAR
REFERENCES I F. HUTCHINSON AND H. B. HALES, J. Mol. Biol., 5 ° (197o) 59. 2 A. WACKER, in J. N. DAVIDSON AND V~T. E. COHN, Progress in Nucleic Aacid Research. Vol. i, 1963, p. 369. 3 W. D. RuPP AND W. H. PRUSOFF, Biochem. Biophys. Res. Commun., 18 (i965) 145. 4 M. F i K u s , K. L. WIERZCHOWSKI AND D. SHUGAR, Biochem. Biophys. Res. Commun., 16 (i964) 478 • 5 M. FIKuS, K. L. WIERZCHOWSKI AND D. SHUGAR, Photochem. Photobiol., 4 (1965) 52I6 H. A. LOZERON, M. P. GORDON, T. GABRIEL, W. TAUTZ AND R. DUSCHINSKY, Biochemistry, 3 (1964) 1844. 7 Z. KAZlMIERCZUK AND D. SHUGAR, Acta Biochim. Polon., 17 (197 o) 325 . 8 R. 1Y[. CRESSWELL AND H. C. S. WooD, J. Chem. Soc., (196o) 4768. 9 T. KULIKOWSKI AND D. SHUGAR, Acta Bioehim. Polon. 18 (1971) 2o9. i o J. J. F o x AND D. SHUGAR, Bull. Soc. Chim. Belg., 61 (1952) 44. I I D. SHUGAR, in E. CHARGAFF AND J. ~NT.DAVIDSON, The Nucleic Acids, Vol. 3, A c a d e m i c Press, N e w Y o r k , I96O, p. 39. 12 I. WEMPEN AND J. J. F o x , J. Am. Chem. Soc., 86 (i964) 2774. 13 I. WEMPEN AND J. J. F o x , jr. Med. Chem., 7 (1966) 207 . 14 C. JANION AND D. SHUGAR, Aeta Biochim. Polon., 7 (196o) 309. 15 P. CERUTTI, Y. KONDO, W. LANDIS AND B. WITKOP, J. Am. Chem. Soc., 9 ° (I968) 77 I.
Biochim. Biophys. Acta, 245 (1971) 157-166
I57
BBA 97073
PHOTOCHEMICAL TRANSFORMATION OF 6-CHLOROURACIL AND SOME A L K Y L A T E D ANALOGUES Z. K A Z I M I E R C Z U K AND D. SHUG-AR
Department o/ Biophysics, Institute o/ Experimental Physics, University o/ Warsaw, and Institute o/Biochemistry and Biophysics, Academy of Sciences, Warsaw (Poland) (Received July 6th, I971)
SUMMARY.
The monoanionic, but not the neutral, form of 6-chlorouracil in aqueous medium undergoes quantitative photochemical transformation to barbituric acid. Similarly, I-methyl-6-chlorouracil and 3-methyl-6-chlorouracil monoanions are transformed to I (3)-methylbarbituric acid, and 5-ethyl-6-chlorouracil to 5-ethylbarbituric acid, all in quantitative yield. The quantum yields for these reactions are quite high, 0.05 to 0.09, and unaltered over the wavelength range 254-280 nm, or b y a Io-fold change of concentration of the initial substances. The rates of photochemical transformation are independent of the O H - concentration, but exhibit a marked deuterium isotope effect. A reaction scheme consistent with the foregoing, and other, data is the photohydration of the 5,6 bond of the excited singlet state of the chlorouracil monoanion, followed b y the very rapid dark elimination of C1- to give barbitufic acid. The findings are discussed in relation to the photochemistry of 5-halogeno analogues of thymine.
INTRODUCTIO N
The photochemical transformations of 5-halogenouracils have been extensively investigated, largely because of the fact that 5-bromouracil and 5-iodouracil are base analogues of thymine and undergo incorporation into cellular or viral DNA, whereas 5-fluorouracil is a base analogue of uracil and can replace the latter in RNA. The replacement of thymine or uracil in a nucleic acid b y a 5-halogenouracil is usually accompanied by a pronounced modification in radiation sensitivity, as estimated by either biological or physicochemical criteria (ref. i, and references cited therein). However, the nature of the photoproducts of the 5-halogenouracils, and the reaction mechanisms involved, have not been satisfactorily clarified. In the case of 5-bromo- and 5-iodouracils, dehalogenation is observed, with partial formation of uracilZ,3; at least in the case of 5-iodouracil the generation of a free radical at the C-5 of the pyrimidine ring is involved ~. Dehalogenation is likewise observed with 5fluorouracil; the reaction in this instance has been shown to be wavelength dependent 4-~. At wavelengths to the red of 270 nm, a unique photoproduct is formed which has been identified as the hydrate, 5-fluoro-5,6-dihydro-6-hydroxyuracil; and this product can be further photochemically transformed b y irradiation at shorter wavelengths to eliminate fluoride anion with simultaneous formation of barbituric acid. Biochim. Biophys. Acta, 254 (197 I) 157-166
I5~
z. KAZIMIERCZUK,D. SHU(;AR
During the course of a study on the specific N-methylation of 6-chlorouracil, the products of which are intermediates in the synthesis of purines and pteridines 7, it was noted that ultraviolet irradiation of aqueous solutions of 6-chlorouracil and its alkylated derivatives led to conversion to new products with relatively high quantum yields. This finding was rather unexpected since, under analogous conditions, 5-chlorouracil is remarkably radiation resistant, being transformed (to unidentified products) with a quantum yield of less than IO-4 (M. FIKUS AND D. S~IUGAR, unpublished observations).
MATERIALSAND METHODS 6-Chlorouracil was prepared according to the method of CRESSWELL AND WOODs, and 5-ethyl-6-chlorouracil as described elsewhere 9. The preparation of 3methyl-6-chlorouracil, the previously unknown I-methyl-6-chlorouracil, and 1,3dimethyl-6-chlorouracil was based on the selective methylation of 6-chlorouraciU. The properties of the samples of 5-ethylbarbituric acid and I(3)-methylbarbituric acid, used as reference compounds and as filters, are described elsewhere 9,1°. All compounds were checked b y melting points and chromatography in several solvent systems. Ascending paper chromatography was employed with W h a t m a n paper No. I and following solvent systems: (A), water-saturated n-butanol; (B) water-saturated n-butanol with ammonia in the gas phase; (C) n-butanol-water-acetic acid (7 : 2 : I,
v/v/v). Phosphate buffers were employed in the alkaline pH range, where maintenance of constant pH was essential. A Radiometer PHM22 compensating pH meter with semi-micro glass electrode was used for controlling pH. Solutions were irradiated in Io-mm or I-mm path length quartz spectrophotometer cuvettes, the latter being employed when irradiated solutions were to be chromatographed, or for following rates at a lo-fold higher concentration, h~radiation involved the u.~e of three types of sources, as follows: (a) A Hanau Q7oo medium pressure mercury lamp with filters as indicated to isolate a given spectral region. (b) A Thermal Syndicate 254 nm mercury resonance lamp, with a sodium acetate filter to eliminate wavelengths below 230 nm. The intensity of this lamp at the cuvette surface was 7.5 " IOle quanta/cm2 per rain at 254 nm. (c) A Jarrell-Ash f I :3-5 grating monochromatgr with a I5o-W Hg-xenon lamp provided with a specially constructed condensing reflector for illumination of the entrance slit. The exit slit was set at 2 ram, corresponding to a band-width of 6 nm. This sorce was used to irradiate solutions in semi-micro cuvettes of width 4 mm, the solution being stirred continuously with a fine wire. Under these conditions the effective average intensity over the solution surface was 3.2 • lO15 quanta/cm 2 per min at 28o nm. Light intensities for sources (b) and (c) were estimated to about 5 % with the aid of a neutral aqueous solution of IO-* ~ uridine, using a quantum yield for uridine hydration of o.o21, known to be independent of the wavelength n. Actinometry was carried out in the same cuvettes used for irradiating the solutions under study. Biochim. Biophys. Acta, 254 (1971) 157-I66
PHOTOCHEMISTRY OF 6-CHLOROURACILS
159
RESULTS
Irradiation of 6-chlorouracil, lO -4 M in aqueous medium, pH approx. 6, at 254 nm led to the gradual disappearance of the characteristic absorption maximum at 283 nm, with the concomitant appearance of a new band centred at about 258 nm. Following irradiation, acidification of the solution to pH 2 with HC1 liquidated the 258 nm band which, however, was restored on neutralization of the solution. This behaviour of the 258 nm band is reminiscent of that for barbituric acid 1°. Paper chromatography of the irradiated solution, using Whatman No. I with various solvent systems (Table 2) demonstrated the presence of starting material and a second spot with an R~. identical with that for an authentic sample of barbituric acid. TABLE
I
ASCENDING RESPONDING
PAPER
CHROMATOGRAPHY
OF SOME DERIVATIVES
BARB1TUEIC ACIDS PHOTOCHEMICALLY
Compound
6-Chlorouracil B a r b i t u r i e acid 3-Methyl-6-chlorouracil i-Methyl-6-chlorouracil i ( 3 ) - M e t h y l b a r b i t u r i c acid 5-Ethyl-6-chl°r°uracil 5 - E t h y l b a r b i t u r i c acid
DERIVED
OF 6 - C H L O R O U R A C I L
AND
THE
COR-
FROM THEM
R F in solvent system A
B
C
o.54 o.12 o.77 o.62 0.49 o.84 0.44
o.63 o.21 o.76 -0.48 o.76 o.36
o.78 o.5o o.83 o.57 0.52 o.83 0.65
Since the pKa of 6-chlorouracilis 5.7 (ref. 12), a solution at pH 6 necessarily includes a mixture of the neutral and monoanionic forms. Irradiation of a solution of 6-chlorouracil at pH values below 6 led to a marked decrease in the rate of photochemical transformation and, at pH values below 4, no barbituric acid could be detected amongst the photoproducts. B y contrast, irradiation in buffered medium at pH values above 7, where 6chlorouracil is virtually in the form of the monoanion, led to an increase in the rate of photolysis, and in the extent of formation of barbituric acid. However, under these conditions, barbituric acid itself is exclusively in the form of the monoanion (pK 3.9, see ref. Io), with an absorption maximum at 258 nm and emax of 21.5" lOs, so that ez54nm is 18 • 103. Consequently the barbituric acid formed during irradiation of 6-chlorouracil is itself subject to photochemical transformation, when irradiated at 254 nm. Separate trials demonstrated that barbituric acid monoanion is considerably more radiation resistant than 6-chlorouracil, with a quantum yield for transformation at least an order of magnitude lower (c/. ref. Ii). But it appeared simpler to eliminate the possible complicating effects of irradiation on the barbituric acid formed, by irradiating at wavelengthts to the red of the long wavelength shoulder of barbituric acid monoanion, i.e. to the red of 275 nm. A lO -4 M solution of 6-chlorouracil in 0.02 M buffer, pH 8.7, was first irradiated with the Hanau Q7oo lamp through a Schott-Jena WG-7 filter cutting off completely at 265 nm. Subsequently this filter was replaced b y a 2-ram layer of 5 " IO-~ IV[ barBiochim. Biophys. Acta, 254 (197 I) I 5 7 - I 6 6
160
Z. KAZIMIERCZUK, D. SHUGAR
bituric acid monoanion (pH about 7)- The optical density of this filter is so high, about 2o A25s nm units, that it effectively cuts out radiation which would normally be adsorbed by barbituric acid formed in the irradiated solution, while the change in transmission during the irradiation times employed is negligible. Under these conditions, the course of photochemical transformation of 6-chlorouracil with time proceeded as depicted in Fig. I. It will be noted, from the two isosbestic points formed at 267.5 and 232 rim, that a single ultraviolet-absorbing photoproduct is formed from 6-chlorouraeil. Paper chromatography did, in fact, confirm the formation of only one ultiaviolet-absorbiug photoproduct (Table I), with RF values corresponding to authentic barbituric acid, further confirmed b y spectral examination of the eluates. That barbituric acid was the only product formed was supported b y a comparison of the optical density at ~.rr~x of the initial solution of 6-chlorouracil to that of the presumed barbituric acid spectrum following complete photolysis of the solution. From Fig. I, the absorbance of the initial 6-chlorouracil solution is o.82, while that of the photoproduct formed after 35 min irradiation is 1.36. The ratio of the latter to the former is 1.66. The emax of 6-chlorouracil monoanion is 12.5" IOa, while A I.l,
1.3
1.2
1.2
1.1
1.1
1.2 0.9
~9 0.8
t(in)
o.t
(11 ~s
s
0~ o3
l0
d (14
~____~0
~3
Q~ k\ o.1
20 s
220 230 2/,0 "250 260 270 280 290 300 3%0 3')0
AInrn)
O~ 220
2O
~o 2~o 2~o 2~o 2~o 280 ~o 300 glo A(nm}
Fig. I. Course of conversion w i t h t i m e of irradiation, w i t h source (a), of IO-1 or lO -8 M 6-chlorouracil m o n o a n i o n in o.o2 M p h o s p h a t e buffer (pH 8.7) to b a r b i t u r i c acid m o n o a n i o n . The radiation w a s filtered t h r o u g h a 2 - m m layer of 5 " I ° - S M b a r b i t u r i c acid monoanion, so t h a t b a r b i t u r i c acid f o r m e d in the reaction e u v e t t e a b s o r b e d no radiation. Figures denote irradiation times in minutes. N o t e isosbestic p o i n t s a t 267. 5 n m a n d 232 nm, p o i n t i n g to q u a n t i t a t i v e f o r m a t i o n of b a r b i t u r i c acid f r o m 6-chlorouracil. Following completion of reaction, solution w a s acidified to pFI 2 to give a b s o r p t i o n s p e c t r u m of n e u t r a l f o r m of b a r b i t u r i c acid (dashed line). Fig. 2. Course of t r a n s f o r m a t i o n of lO -41V[ 3-methyl-6-chlorouracil m o n o a n i o n in o.o2 M phosp h a t e buffer (pH 8.7), to I ( 3 ) - m e t h y l b a r b i t u r i c acid b y irradiation w i t h source (a), Q-7oo merc u r y lamp, t h r o u g h a 2 - m m layer of 5 " lO-8 IV[ I ( 3 ) - m e t h y l b a r b i t u r i c acid m o n o a n i o n . F i g u r e s denote irradiation t i m e s in rain. Isosbestic p o i n t s at 268 n m and 232 n m point to f o r m a t i o n of only one u l t r a v i o l e t - a b s o r b i n g p h o t o p r o d u c t , I ( 3 ) - m e t h y l b a r b i t u r i c acid.
Biochim. Biophys. Acta, 254 (1971) 157-166
PHOTOCHEMISTRY OF 6-CHLOROURACILS
161
that for barbituric acid monoanion is 21.5 • IOa (ref. IO), the resulting ratio being 1.72. The agreement is seen to be excellent. Analogous results were obtained on irradiation, under similar conditions, of 3-methyl-6-chlorouracil (Fig. 2) and 5-ethyl-6-chlorouracil (Fig. 3), both at appropriate pH values to give the monoanionic forms. The former was irradiated through a (I)3-methyl barbituric acid monoanion filter, which provides reasonably good protection of the I (3)-methylbarbituric acid expected as photoproduct. The latter was irradiated through a filter of 5-ethylbarbituric acid to protect the 5-ethylbarbituric acid expected from photolysis of 5-ethyl-6-chlorouracil. From Fig. 2 it will be observed that the absorbance of the initial 3-methyl-6chlorouracil is 0.70, while that of the product formed is 1.o7, giving a ratio of 1.53. The extinction coefficients at 2max of 3-methyl-6-chlorouracil monoanion and 3 (I)methylbarbituric acid are 12.4 and 20. 3 (refs. 12 and IO), respectively, so that their ratio is 1.64, in reasonable agreement with the observed value of 1.53, and again testifying to quantitative transformation of 3-methyl-6-chlorouracil to 3(I)-methyl barbituric acid. From Fig. 3, the observed ratio of absorbances at the maxima of the initial 5-ethyl-6-chlorouracil and the photoproduct formed following its disappearance is 1.71. This is to be compared with the ratio of the extinction coefficients at 2max of A
A
t.1
1.0
O.9
Q~
0.6
0.7
Qsj (1.5
~ ~4
25 75
o~-
\.\
~ / / ,
,
\\\
0.2
i
220 ~30 240 2gO 260 270 280 "290 300 310 a(nrnl
"220 "230 2~0 2S0 260 270 280 290 300 3t0 3"20 ~(.m)
Fig. 3. Course of t r a n s f o r m a t i o n of lO -41Vf 5-ethyl-6-chlorouracil m o n o a n i o n in 0.02 ]Vf p h o s p h a t e buffer (pH 8.7) to 5-ethylbarbituric acid m o n o a n i o n b y irradiation w i t h source (a) t h r o u g h a 2 - m m layer of 5 " lO-S ]Vf 5 - e t h y l b a r b i t u r i c acid monoanion. Figures denote irradiation times in min. Isosbestic p o i n t s at 279 n m and 235 n m p o i n t to f o r m a t i o n of only one ultravioleta b s o r b i n g product, 5 - e t h y l b a r b i t u r i c acid. Fig. 4. Course of conversion of lO -41Vf I-methyl-6-chlorouracil m o n o a n i o n in o.oi ]Y[ N a O H (pH a b o u t x2) to I ( 3 ) - m e t h y l b a r b i t u r i c acid b y irradiation w i t h source (a) t h r o u g h a 2-ram layer of 5 " 10 -3 M of i - m e t h y l b a r b i t u r i c acid monoanion. Figures denote irradiation time in min. Note isosbestic p o i n t s at 268 n m and 238 nm, p o i n t i n g to f o r m a t i o n of only one u l t r a v i o l e t - a b s o r b i n g p h o t o p r o d u c t , i - m e t h y l b a r b i t u r i c acid. Following 75 rain irradiation, b r e a k a w a y from t h e isosb e s t i c p o i n t occurred due to p h o t o l y s i s of p h o t o p r o d u c t . Hence at this p o i n t the solution was acidified to p H 2 to liquidate t h e a b s o r p t i o n s p e c t r u m of the I - m e t h y l b a r b i t u r i c acid p h o t o p r o duct, a n d t h u s reveal the s p e c t r u m of the unreacted I-methyl-6-chlorouracil (neutral form), w h i c h was e s t i m a t e d q u a n t i t a t i v e l y from its a b s o r b a n c e and k n o w n extinction coefficient.
Biochim. Biophys. Acta, 254 (1971) 157-166
162
z. KAZIMIERCZUK, D. SHUGAR
5-ethyl-6-ehlorouraeil (11. 9 • lO s, see ref. 9) and 5-ethylbarbituric acid (21 • io ~, see ref. 9), which is 1.77. For I-methyl-6-chlorouracil the same photochemical transformation was found, but in this instance the observations were somewhat complicated b y the fact that the absorption spectrum of the monoanion closely overlaps that of the product, I-methylbarbituric acid monoanion (Fig. 4)- Irradiation of I-methyl-6-ehlorouracil monoanion at p H 12 (o.oi M NaOH, pK of starting product 9.05) through a I-methylbarbituric acid monoanion filter proceeded with quantitative conversion to I-methylbarbituric acid only to the point where about 60 0/o conversion had occurred. Following this, a breakaway from the isosbestic point at 268 nm demonstrated the simultaneous photolysis of the i-methylbarbituric acid monoanion product in the reaction mixture. Consequently, following photolysis to the point where d e p a r t m e from the isosbestic point occurred, the solution was acidified to p H 2 to liquidate the spectrum of I-methylbarbituric acid monoanion, and to simultaneously place in evidence the spectrum at this p H of the neutral form of the remaining I-methyl-6-ehlorouracil (see Fig. 4). Subsequent calculation from the spectra of the amount of I-metbyl-6chlorouracil which had disappeared after 60 °/o photolysis, and the amount of barbituric acid formed, using their known extinction coefficients, demonstrated a difference between the two of only 7 9/o• For all four 6-chlorouracil monoanion derivatives, the rate and course of photochemical transformation was unaltered when irradiation was conducted at concentrations of lO -4 M (Io-mm cuvette) and lO -3 M (I-ram cuvette), the cuvettes being suitably positioned with regard to the source so that the incident intensities were the same. The reaction rates and products were also unchanged in the presence of air or nitrogen, the latter of which was flushed through the solutions prior to, and during irradiation. The liberation of CI-, which would be anticipated from the conversion of ehlorouracil to barbituric acid, was verified qualitatively during the course of the reactions b y periodic additions of AgNOs to precipitate AgC1. No attempt was made to quantitatively estimate C1-formation in view of the quantitative character" of the spectral data and confirmation of these b y chromatography. Liberation of C1- as the anion was further confirmed b y the observation that irradiation in unbuffered medium led to acidification during the course of the reaction; when the initial pH was uot for removed from the pK, the rate of photochemical transformation progressively decreased during irradiation. By contrast, when the reactions were conducted in buffered medium, at buffer concentrations sufficient to counteract the C1- liberated, reaction rates were found to be relatively unaffected b y an increase in the p H of the medium for p H values well above ( > I p H unit) the p H of the irradiated derivative, i.e. the reaction rate was not dependent on the O H - concentration.
Quantum yields With the aid of sources (b) and (c), measurements of quantum yields were carried out at 254 and 280 nm b y following quantitatively b y spectral methods the disappearance of starting product and appearance of the appropriate barbituric acid photoproduct, for low degrees of photolysis. The measured values of q~ are listed in Biochim, Biophys. Acta, 254 (1971) 157-166
163
PHOTOCHEMISTRY OF 6-CHLOROURACILS
Table II. For I-methyl-6-ehlorouracil, the intensity of the 28o-nm source was too low to permit of a n y reasonable estimation of qs. However, it is of interest that for the other three 6-chlorouracil derivatives, the quantum yields are essentially similar at the two wavelengths. This is further supported b y an examination of Figs. I, 2 and 3, where irradiation made use of a broad band of wavelengths from the filtered source (a). It will be noted that the relative rates of photolysis of 6-chlorouracil, 3-methyl-6chlorouracil and 5-ethyl-6-chlorouracil follow the same order as the quantum yields given in Table II.
TABLE II QUANTUM
YIELDS
RESPONDING
FOR
TRANSFORMATION
BARBITURIC
Compound
6-Chlorouracil i-Methyl-6-chlorouracil 3-Methy 1-6-chlorouracil I, 3-Dimethyl-6-chlorouracil 5-Ethyl-6-chlorouracil a b e a
OF 6-CHLOROURACIL
MONOANION
DERIVATIVES
TO C O R -
ACID MONOANIONS IN AQUEOUS MEDIUM
pK
5.75 9.05 5.95 -6.5
p H o[ irradiated solution
¢s 254 nm
280 nm
8.7b 12.o 8.7b 6. 5
0.060 0.060 o.o44 o.oi 0.090
0.075 -0.055 -0.o9o
8.7°
Product
Barbituric acid I (3)-Methylbarbituric acid i (3)-Methylbarburic acid __e
5 - E t h y l b a r b i t u r i c acid
Values to a b o u t 4 - i o %. Q u a n t u m yield essentially unaltered over p H range 7.8-i 1.3. Unidentified, b u t n o t 1,3-dimethylbarbituric acid. No change in q u a n t u m yield over p H range 7.8-12.
Kinetics and deuterium isotope e/Ject The photochemical conversion, or dechlorination, of the various 6-chlorouracils to the corresponding barbituric acids, since it is independent of the O H - concentration, probably involves a reaction with water and would therefore be expected to exhibit pseudo first-order kinetics. This is supported b y the fact that the reaction rates are unchanged over a Io-fold range in concentrations. Unfortunately it proved difficult to examine directly the reaction order, since the 254-nm source was in the wavelength range where absorption spectra of starting material and product overlap; while at 280 nm the intensity of the monochromator at our disposal was such t h a t inordinately long exposure times would have been required. If, however, the reaction does involve water, an isotope effect might be anticipated. Fig. 5 exhibits the course of disappearance of 6-chlorouracil with source (c) and a barbituric acid filter, when the reaction was run at p H 8.7 in H20 and p2H 8.7 in 2H20. It will be noted t h a t the rate of disappearance of 6-chlorouracil is appreciably slower in 2H20; if w e t a k e the slopes of these curves at zero time, then KH,o/ K2H,O = 2.1. When the reactions were run with source Co), 254 rim, the corresponding isotope effects for 6-chlorouracil, 3-methyl-6-chlorouracil and 5-ethyl-6-chlorouracil were 1.8, 1.85, and 1.8, respectively. Biochim. Biophys. Acta, 254 (1971) 157-166
164
Z. KAZIMIERCZUK, I). 5;H1T(;.~1{
,4
0.5
~4
H
0.3
2
0
~
0.~
0.1
1
2
3
4
5
6
7 8 t (rain)
9
10 1t
12 13
Fig. 5- D e u t e r i u m isotope effect for p h o t o c h e m i c a l conversion of 6-chlorouracil m o n o a n i o n to b a r b i t u r i e acid m o n o a n i o n as m e a s u r e d b y course of disappearance of i • lO -4 M 6-chlorouracil in HzO (at p H 8.7) and in 2H~O (at p i H 8.7), on irradiation w i t h source (a) t h r o u g h a filter consisting of a 2 - m m layer of 5 " lO-8 M b a r b i t u r i c acid monoanion. R a t e of disappearance m e a s u r e d b y decrease in a b s o r b a n c e at Jtmax. I r r a d i a t i o n w i t h source (b), 254 rim, gave similar results for relative rates in H~O and 2H20.
DISCUSSION
Before considering the possible mechanisms for the photochemical dechlorination of the various 6-chlorouracils, it should be noted that the ground state reactive monoanionic forms are not identical for all of them, but are divided into two groups, as in Scheme I(see refs. 7, 12). O
O
~ N ~ R 1
:
H R I = H~ R2=CH 3
O
CI
O
N
I
CI
Or" 1~1= CH2CH3,R2~-H or RI:
R2= 1-1
CH 3
Scheme I
If we recall (Table I I ) that, for each compound, the rate of photochemical transformation is essentially unaffected b y an increase in p H (in the p H range in which the reactive monoanionie forms exist), this implies that the photoreaction rate is independent of the O H - concentration. It is of interest in this connection that all the chlorouracil analogues exhibit remarkable stability in the ground state, even in hot acid and alkali 13. A plausible mechanism for the photochemical dechlorination reaction m a y therefore be presented as in Scheme 2 for the case of 6-chlorouracil, and involving hydration of the 5,6 bond, followed b y elimination of HC]. We have, of course, no direct evidence for the presumed hydrated intermediate, shown in brackets. Biochim. Biophys..4cta, 254 (1971) 157-166
PHOTOCHEMISTRY OF 6-CHLOROURACILS
165
o
H N( + I-t2o ©
ci
---
{
H
OH Cl
~
/H
___
-HCt O"
"N"
"O
I
H
Scheme 2
The absence of an oxygen effect m a y be taken to imply that the presumed hydration proceeds via the excited singlet state of the 6-chlorouracil monoanion, in accordance with the photohydration of uracil and cytosine, which are generally accepted as involving the excited singlets. It remains to establish whether the elimination of the C1- monoanion is photochemically induced, as in the case of hydrated 5-fluorouracil, which is stable at neutral p H but photochemically eliminates F - on irradiation at 254 nm to also give barbituric acid a. The data in Table I I provide an apparently unequivocal answer to this question. The presumed hydrated intermediate in Scheme 2 should, like hydrated 5-fluorouracil 4 or 2,4-diketo-5,6-dihydropyrimidines in general 14, exhibit low or negligible ultraviolet absorption at 280 nm relative to 254 rim. Notwithstanding this, the q u a n t u m yields at 280 nm, for those three compounds for which measurements were possible, are not inferior to those at 254 nm (Table II). It follows that the elimination of C1- from the presumed hydrated intermediate must be a dark reaction which is extremely rapid, so that the values given in Table I I must represent quant u m yields for the hydration reaction. This is indirectly supported b y the observed deuterium isotope effect for the overall reaction in 2H20 , b y analogy with the corresponding deuterium isotope effects foi the photohydration of the 5,6 bonds of uracil n and 5-fluorouracil analogues 4. It is of supplementary interest that, in contrast to the 6-chlorouracil derivatives embraced in this investigation, 5-fluorouracil will readily undergo hydration either in the neutral or the monoanionic form 5. The extreme instability of the presumed hydrated intermediate shown in Scheme 2 is in agreement with the fact that 5,6-dihydroderivatives of 6-halogenouracils are unknown, whereas those of 5-halogenouracils are readily prepared. An a t t e m p t was actually made to obtain the 5,6-dihydro derivatives of 6-chlorouracil b y photochemical reduction with K B H 4 as described b y CERUTTI et al. 15 for photochemical reduction of uracil ring. The results were completely negative, the only consequence being the reaction of K B H 4 with the barbituric acid photoproduct. Finally, we should like to emphasize that, apart from the interest of the foregoing reactions in relation to the photochemical behaviour of 5-halogenouracils, the photochemical dechlorination reactions herein described are quite unusual in themselves; and, from a perusal of the literature, are conspicuous by their absence. ACKNOWLEDGMENTS
We are indebted to Mgr. T. Kulikowski for the samples of 5-ethyl-6-chlorouracil and 5-ethylbarbituric acid; to Dr. Ewa Sztumpf-Kulikowskafor discussions; and to Mgr. K. Berens for assistance with the source reflector for the monochromator. This investigation was supported in part b y the World Health Organization, The Wellcome Trust, and the Agricultural Research Service, U.S. Department of Agriculture. Biochim. Biophys. Acta, 254 (1971) 157-166
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z. KAZIMIERCZUK, D. SHUGAR
REFERENCES I F. HUTCHINSON AND H. B. HALES, J. Mol. Biol., 5 ° (197o) 59. 2 A. WACKER, in J. N. DAVIDSON AND V~T. E. COHN, Progress in Nucleic Aacid Research. Vol. i, 1963, p. 369. 3 W. D. RuPP AND W. H. PRUSOFF, Biochem. Biophys. Res. Commun., 18 (i965) 145. 4 M. F i K u s , K. L. WIERZCHOWSKI AND D. SHUGAR, Biochem. Biophys. Res. Commun., 16 (i964) 478 • 5 M. FIKuS, K. L. WIERZCHOWSKI AND D. SHUGAR, Photochem. Photobiol., 4 (1965) 52I6 H. A. LOZERON, M. P. GORDON, T. GABRIEL, W. TAUTZ AND R. DUSCHINSKY, Biochemistry, 3 (1964) 1844. 7 Z. KAZlMIERCZUK AND D. SHUGAR, Acta Biochim. Polon., 17 (197 o) 325 . 8 R. 1Y[. CRESSWELL AND H. C. S. WooD, J. Chem. Soc., (196o) 4768. 9 T. KULIKOWSKI AND D. SHUGAR, Acta Bioehim. Polon. 18 (1971) 2o9. i o J. J. F o x AND D. SHUGAR, Bull. Soc. Chim. Belg., 61 (1952) 44. I I D. SHUGAR, in E. CHARGAFF AND J. ~NT.DAVIDSON, The Nucleic Acids, Vol. 3, A c a d e m i c Press, N e w Y o r k , I96O, p. 39. 12 I. WEMPEN AND J. J. F o x , J. Am. Chem. Soc., 86 (i964) 2774. 13 I. WEMPEN AND J. J. F o x , jr. Med. Chem., 7 (1966) 207 . 14 C. JANION AND D. SHUGAR, Aeta Biochim. Polon., 7 (196o) 309. 15 P. CERUTTI, Y. KONDO, W. LANDIS AND B. WITKOP, J. Am. Chem. Soc., 9 ° (I968) 77 I.
Biochim. Biophys. Acta, 245 (1971) 157-166