R. M. DANZIGER, E. HAYON,AND Ad, E. LANGMUIR
3842
Pulse-Radiolysis and Flash-Photolysis Study of Aqueous Solutions
of Simple Pyrimidines. Uracil and Bromouracil
by R. M. Danziger, E. Hayon, and M. E. Langmuir Pioneering Research Laboratory, U.S. A r m y il’atick Laboratories, Natick, Massachusetts (Received M a y 7 , 1968)
01 760
The pulse radiolysis of uracil and 5-bromouracil has been studied at pH 1.0, 5.5, and 11.0 in argon-, air- and N20-saturated aqueous solutions. The main precursor which gives rise to the observed transient optical absorption spectra in these systems was the OH radical. The transient species, which decay according to the second-order rate law, are considered to be formed from the addition of OH radicals to the 5,6 double bond of the pyrimidine molecule. In the flash photolysis of neutral and acidic solutions of 5-bromouracil, at 320 nm and 420 nm which decay by second-order processes. two major transients are produced with A, One of the main primary photolytic processes is homolytic cleavage of the C-Br bond to produce Br radicals, as evidenced by the formation of Brz- radical anions on photolysis in the presence of bromide ions. Photolysis of the bromouracil anion (RO-) in air-freealkaline solution produces two major species with absorption maxima at 330 and 420 nm. The “330” transient appears to result from the photoioniaation of RO- to RO. e&q-. A similar RO . transient species is formed in the pulse radiolysis of alkaline air-free aqueous solutions of ROions, produced by abstraction of an electron from RO- by OH radicals.
+
Introduction Radiation chemical studies of some simple pyrimidines such as uracil, thymine, and cytosine in dilute aqueous solutions have shown’-7 that OH radicals formed in the radiolytic decomposition of water add preferentially to the 5,6 carbon-carbon double bond to form the hydroxypyrimidyl radical. I n oxygenated solution, this radical is scavenged by oxygen and eventually yields the 5,6 glycol as a major radiolysis product.’I2 I n a recent investigation on the X irradiation of aqueous solutions of uracil-2-14C, Smith and Hayss have identified the following compounds: dihydrouracil, 6-hydroxy-5-hydrouracil (under Nt, not 0 2 atmosphere), isobarbituric acid, cis- and trans-glycols of uracil, and alloxan. A qualitative and quantitative lack of agreement exists in the data from the different laboratories, due probably to the dependence of G values on pH, oxygen, solute concentration, and impurities. The photochemistry of aqueous solutions of uracil and bromouracil has been studied by a number of investig a t o r ~ . ~ I-n~ ~some of the studies the excitation wavelength was not clearly defined, and the effect of oxygen on the nature of the products was not established. Addition of water to the 5,6 doub1e)ond was found to be induced by uv radiation at 2537 A, and the hydration product has been reported to be 50% reversible in uracil and irreversible in 5-bromouracil on acidification and heating. 9 , 1 3 Dimers have been detected in the photolysis at room temperature of aqueous solutions of uracil’3 and bromouracil.” I n the case of bromouracil, the dimer has been identified’l as 5,s’diuracil. S o conclusive evidence has yet been obT h e Journal of Physical Chemistry
tained for the participation of triplet states in the photochemistry of uracil or bromouracil in aqueous solutions, and the nature of the primary photolytic processes which give rise to the observed chemical products is still undefined. I n view of the above situation, a preliminary study of the radiation chemistry and photochemistry of aqueous solutions of uracil and bromouracil has been initiated, using the f ast-reaction techniques of pulse radiolysis and flash photolysis.
Experimental Section Pulse-Radiolysis Setup. The Natick 24-MeV Varian linear accelerator has been used in this work, and full (1) G. Scholes, J. F. Ward, and J. Weiss, J . Mol. Biol., 2, 379 (1960). (2) (a) B. EBert and R. Monier, Nature, 188, 309 (1960);
(b) R. Latarjet, B. Ekert, and P. Demerseman, Radiation Res. Suppl., 3, 247 (1963). (3) A. Kamal and W.
Garrison, Nature, 206, 1315 (1965); J. Holian and W. M. Garrison, Radiation Res., 27, 527 (1966). (4) G. Scholes, Progr. Biophys., 13, 59 (1963). (5) J. Weiss, Progr. Nucleic Acid Res. Mol. Biol., 3, 103 (1964). (6) H. Reuschl, 2. Naturforsch., 21b, 643 (1966). (7) E. Gilbert, 0. Volkert, and D. Schulte-Frohlinde, ibid., 22b, 477 (1967); 0. Volkert, W. Bors, and D. Schulte-Frohlinde, ibid., 22b, 480 (1967). (8) K. C. Smith and J. E. Hays, Radiation Res., 33, 129 (1968). (9) A. D. McLaren and D. Shugar, “Photochemistry of Proteins and Nucleic Acids,” Pergamon Press, Oxford, 1964, p 162. (10) K. C. Smith, Radiation Res. S u p p l . , 6,54 (1966). (11) H. Ishihara and S. Y . TVang, Nature, 210, 1222 (1966); Biochemistry, 5 , 2307 (1966). (12) W.Rothman and D. R. Kearns, Photochem. Photobiol., 6, 775 (1967). (13) A. A. Lamola and J. P. Mittal, Science, 154, 1560 (1966). (14) R. B. Setlow, ibid., 153, 379 (1966).
PULSE RADIOLYSIS AND FLASH PHOTOLYSIS OF PYRIMIDINES details of the experimental conditions will be published elsewhere.16 Briefly, the Linac was operated at about 7 MeV, 200-500 mA current, providing single pulses of electron of 1-2-psec duration. The total dose, measured using 2 x M KBr solutions and ~g,,-~~’’ = 7.8 X lo3M-’ cm-1,16was about 8 X 1017eV/ml per pulse in a rectangular optical cell with dimensions of 30 mm (path length) X 15 X 15 mm. Due to small fluctuations in the dose per pulse, the monitoring light beam was split in two and each beam was allowed to enter a monochromator and photomultiplier unit assembly. Two EM1 9558 QB photomultiplier tubes were employed throughout this work. One monochromator was kept at a fixed wavelength, to act as an internal dosimeter, and was used to normalize the fluctuation in output of the accelerator. A high-pressure xenon arc lamp (Osram XBO 450 W) operated from a highly current-regulated power supply was used as the monitoring light source. Flash-Photolysis Setup. The flash-photolysis lamps and experimental layout used in this work have been described e1~ewhere.l~ General Procedures. The optical absorption spectra of the transient species produced were obtained by the point-by-point method. Reaction rate constants were determined using a computer by least-squares approximation of first- and second-order reaction^.'^'^' Solutions were prepared using water purified by triple distillation, radiolysis, and photolysis.l* Reagents were the best available supplied by Calbiochem Co. and Baker and Adamson. The pH’s were adjusted using perchloric acid and sodium hydroxide.
Results and Discussion Pulse Radiolysis of Uracil. In the pulse radiolysis of oxygen-free aqueous solutions of 2 X lo-* M uracil (U) at pII 5.5 a transient optical absorption spectrum is observed which absorbs in the wavelength range 280500 nm; see Figure lb. This species has two absorption maxima at -400 and -300 nm, and its disappearance follows the second-order rate law, as shown in Table I. A similar absorption spectrum is obtained on pulee radiolysis of deaerated uracil solutions at pH 1.3, Figure l a . The radiation chemistry of water and aqueous solutions is known to produce the reactive intermediates e-, H, and OH radicals, in addition to the “molecular” products Hz and H2Oz
HzO-+ e,,-, H, OH, Hz, and Hz02
(1)
The following reactions can be considered to take place ea,-
eaq-
+U
+
(U-)
+ NzO Kz + OH + OHOH + U + (U*OH) -+
3843
.040
,020 0 -060
7 -
.040
d
Q
.020
o ,000
0.I 5
,060 0.10 .040 0.05
.020
0
300
350
400
450
500
550
0
X ,nm Figure 1. Optical absorption spectra of transient species produced in the pulse radiolysis of aqueous solutions of 2 X 10-4 M uracil in: (a) acid, p H 1.0; (b) neutral, pH 5.5; and (e) alkaline, p H 11.0. Solutions were saturated with argon (O), NzO ( O ) , and air ( 0 ) ; 1-1.5-psec pulses of 7-MeV electrons were used, and OD’s were measured 4 psec after pulse.
with kz = 7.7 X lo9 M-’ sec-I, k~ = 5.6 X log M-l sec-l, and kq = 3.5 X lo9 &f-l sec-l.l9 On pulse radiolysis of N20-saturated uracil solutions, a twofold increase in the 400-nm transient species was observed (Figure 1b) compared to argon-saturated solutions. Since G(e,,-) G(OH), and under the experimental conditions used ( 2 X M U and 2 X M N20) all the ea,- would react with N20, the species with A, -400 nm can be attributed to the reaction of OH radicals with uracil. Based on calculations of the electronic structure of the pyrimidine molecule,20the 5,6 carbon-carbon double bond has been shown to be the most reactive site of the pyrimidine molecule. Hence, it is suggested that the
-
(15) E. D. Black and E. Hayon, to be published. (16) M. S. Matheson, W. A. Mulac, J. L. Weeks, and J. Rabani, J. P h y s . Chem., 70, 2092 (1966). (17) (a) L. Dogliotti and E. Hayon, ibid., 71, 2611 (1967); (b) M. E. Langmuir and E. Hayon, ibid., 71, 3808 (1967).
(3)
(18) E. Hayon, Trans. Faraday Soc., 6 0 , 1059 (1964). (19) M .Anbar and P. Neta, Intern. J . A p p l . Radiation Isotopes, 18, 493 (1967).
(4)
(20) B. Pullman and A. Pullman, “Quantum Biochemistry,” Interscience Publishers, New York, N. Y . , 1963.
(2)
Volume 78. Number 11 October 1968
R. Ad. DANZIGER, E. HAYON, AND NI. E. LANGMUIR Table I : Second-Order Decay Rates of Transient Species Produced in M Uracil the Pulse Radiolysis of Aqueous Solutions of 2 X Absolute rate, M-1 sec-I
System
X , nm
1.3 1.3 1.3
Argon-saturated Argon-saturated Argon-saturated
305 340 390
5 . 6 f 1 . 3 X lo6 1 . 3 0 . 3 X IO6 1 . 6 f 0 . 4 X lo6
5.5 5.5 5.5 5.5
Argon-saturated Argon-saturated N20-saturated NzO-saturated
310 410 310 400
1 . 3 f 0 . 4 X lo6 1 . 8 f 0 . 4 X lo6 2 . 2 1 0 . 5 x 106 1 . 3 f 0 . 3 X lo6
11.o 11.o 11.0 11.o 11.o
Argon-saturated Argon-saturated Argon-saturated NzO-saturated N2O-saturated
320 400 400 390 390
1.11 0.2 x 8 . 6 =t1.8 X 3.0 1 1.0 X 8.6 f 2.2 X 3 . 2 f 0.8 X
PH
’
2k/r
106 lo6‘ lo6“ lob*
x
9.0
loEd
...
... 2.1 x 1 0 9 ~ 1.8 x 1 0 9 ~ ..
... 8.2 x 109’ ...
... x 109~
2.2
..
a Fast-decaying transient. Slow-decaying transient. Deviation 1 1 5 % . Based on P O = 1.9 X I O 3 M-1 cm-1. Based on e3O0 = 7.5 X lo3 M - l cm-l. ‘ Based on €390 = 2.6 x IO3 M-1 cm-1. on e4O0 = 1.0 X l o 3 M-I crn-l.
’
‘400-nm” transient is the result of OH-radical addition to the 5 or 6 position 0
H
0
0
Support for reaction 4’ can be derived from the observed formation of pyrimidine glycols and of hydroxy-hydro corn pound^.^^^ Due to the irreproducibility in the formation of the 300-nm species in the pulse radiolysis of neutral air-free solutions, it is not possible to establish its radical precursor. Uracil is very reactive toward eaq- with IC2 = 7.7 X lo9 sec-l, but no intermediate has been observed within the time resolution of this work ( 3 1-2 psec) in the radiolysis of neutral air-free solutions, which can be definitely attributed to such a reaction. In alkaline solution, however, a strong absorbing species is produced, with Amax-320nm, on pulse radiolysis of oxygen-free 2 X uracil at pH 11.0, as shown in Figure IC. In XZOsaturated solution, at pH 11.0, this transient absorption at 320 nm is not observed. Consequently, it is tentatively suggested that the 320-nm transient is the mononegative uracil radical anion (U-). This species is not observed at lower pH’s, due possibly to the protonation of U-. Further work is in progress to establish this point. Two other transient species are produced in the pulse radiolysis of Ar- or NzO-saturated uracil solutions at The Journal of PhVsical Chemistry
e
Based
pH 11.0. One species has a maximum a t A,, -400 nm and is shown in Figure IC. The other transient species decays more slowly than the Amax 400-nm species and appears to have a rather broad band in the region 400-580 nm. Both species decay by second-order processes, and their decay rates are given in Table I. A permanent product(s) which absorbs at wavelengths below 360 nm is also produced in NZO- and Ar-saturated solutions of uracil at pH 11.0. The first pK of uracil due to dissociation at the Cz position is at pK = 9.5.21 At pH 11.0 one could therefore expect different sites of radical attack on the uracil anion, RO-, compared to its un-ionized form present in neutral solutions. It) is suggested that a t pH 11.0 the OH radicals can add to the 6,6 double bond or abstract an electron from the Cz-carbonyl to produce an RO radical OH
+ RO-
+RO*
+ OH-
(5)
In the puke radiolysis of neutral and acidic solutions of uracil in the presence of oxygen, a very short-lived intermediate with a lifetime of
