489
PHOTOREACTIONS IN AQUEOUS SOLUTIONS OF THYMINE, pH 12 15%. The ketone conversion was generally about 3%. Adopting’ ICz == 2.3 X l O I 3 cm8 mol-’ sec-I, we obtain k3 = 5.2 X 1O’O exp(-3900/RT) cm3 molw1 sec-’, which may be compared3 to lei = 3.9 X 1O1O exp(-3900/RT) em3 mol-’ sec-l. The similarity in the Arrhenius parameters suggests that reaction 3 occurs predominantly by addition to the N atom.* Because of the rapid addition reaction, the competition between abstraction and combination (reactions 5 and 2) is not easily studied. However, for the small yields of CF3Hobtained, we may write k5/k2”2
=
Rc~~H/Rc~F~~’~[imine] (8)
The Arrhenius plot for expression 8 is shown graphically in Figure 1, from which a value of kg = 1.6 X lo9 exp(-4500/RT)cm3 mol-’ see-’ is obtained. The low activation energy is compatible with similar studies involving >N-H group^,^ but the A factor appears to be somewhat lower than that for comparable systems. However the agreement between ka/ks obtained in this work and that obtained in the direct photolysis of the imine2lends credence to our value.
The data are consistent with the production of CFa radicals in the direct photolysis of the imine. At low stationary CFB radical concentrations CzFB production would not occur due to the highly efficient addition reaction. The formation of CF3H in the direct photolysis may be attributed, in part, to H-atom abstraction by CF8 radicals as well as to an intramolecular elimination from the photoexcited imine. The primary processes are discussed in detail elsewhereq2 Acknowledgment. D. W. F. and G. 0. P. thank the Clapp Foundation, Milford, Conn., for generous support, and s. T. thanks Rutgers University for a Faculty Fellowship. The partial support of the National Science Foundation is also acknowledged. (7) P. B. Aysoough, J . Chem. Phys., 24, 944 (1956). (8) The data at 154’ give log kz/k3*/2 values of 2.07 and 2.25, respectively, which lie close to the line in Figure 1. However, they are
omitted due to the low CzFa yields and some interference by about equivalent amounts of COZin the analysis. A trace (-0.025%) of COz was present in the HFA, but its formation in the reactor at 184’ could occur to a slight extent. See ref 3. (9) P. Gray, A. A. Herod, and A. Jones, Chem. Rev., 71, 247 (1971).
Photoreactions in Aqueous Solutions of Thymine, pH 12 by D. W. Whillansl and H. E. Johns* Department of Medical Bioph,hysics, University of Toronto, and The Ontario Cancer Institute, Toronto, Ontario, Canada Publication costs assisted by The Ontario Cancer Institute
Aqueous solutions of thymine a t pH 12 have been studied by flash photolysis. Under these conditions we see a transient species due to the hydrated electron which arises from excitation of OH-. This electron absorption is quenched by NzO, by 0 2 a t a rate of 1.6 X 1O1O M-l sec‘l and by the thymine anion a t a rate of 3.0 x log M-I sec-1. I n addition two species which we attribute to absorption by thymine alone are observed. One of these is short-lived and quenched by 0 2 ; it is also quenched by ground-state thymine a t a rate of 7.3 X 108 M-I sec-I and is almost certainly the triplet state. The spectrum of this species is red-shifted 60 nm compared to that of the neutral triplet seen a t pH 7. The other species produced in thymine alone has a lifetime of at least 1 msec and may in fact be a stable product. This species is not quenched by 02. I t s nature is unclear.
Introduction When neutral aqueous solutions of thymine are irradiated with ultraviolet (uv) light, permanent photodimers identical with those which cause biological inactivation of DNA are produced.2 These dimers have been shown to result from the bimolecular reaction of a thymine ground state and an excited triplet state. The triplet state has been detected by flash photolysis techniques and has been characterized by its spectrum. Rate constants for reactions with ground-state thymine and 0 2 have been determined.3
Our interest in the excited states of thymine led us to examine species produced in solutions of thymine above the pK for ring ionization which occurs about p H 9.9.4 Changes in properties of the excited states a t high pH are not unexpected since one tautomeric (1) Research Fellow, National Cancer Institute of Canada.
(2) G. J. Fisher and H. E. Johns, Photochem. Photobiol., 11, 429 (1970). (3) D. W. Whillans and H. E. Johns, J . Amer. Chem. Soc., 93, 1368 (1971). (4) D. Shugar and J. J. Fox, Biophys. Biochem. Acta, 9, 199 (1952).
The Journal of Physical Chemistry, Vol. 76,No. 4, 1878
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D. W. WHILLANS AND H. E. JOHNS
form of thymine has been shown to fluoresce strongly in such solutions whereas thymine fluoresces only very weakly a t neutral P H . ~ We also expect that bimolecular reactions between the triplet and ground states should be inhibited by mutual repulsion of the negative species. Indeed, dimer photoproducts have not been detected in alkali solutions, although their instability in such solutions may have made their detection impossiblee6 At high pH, however, other photolytic reactions occur in aqueous solution. Hart' and others*,@have shown that large yields of eaq- and .OH are produced by reaction 1. The subsequent reactions of these prod-
OH-
+ hv
1.00
1'
I
(left scale)
+
eaq- .OH (1) uct species with thymine, which have so far been studied solely by pulse radiolysis techniques,lO,llare expected in flash systems. The purpose of this paper is to describe these reactions and to characterize the species resulting from the excitation of thymine.
Experimental Section Materials and Methods. Our flash photolysis techniques have already been described,12 and a more detailed account is in ~ r e p a r a t i 0 n . l ~Briefly, the flash results from the discharge of 250 J through two lamps in series, each of which is a hollow quartz cylinder nearly filled by a solid quartz rod at an air pressure of 10 Torr. These lamps are placed on either side of the reaction cell and enclosed in a polished double-elliptical cavity. The uv flux below 320 nm through the cell is about 1 peinstein, as determined by MGL ~a1ibration.l~The spectrum of the flash is limited to about 200 nm by the transmission of the quartz lamps (G.E. Type 204 fused quartz). This is 20 nm below the limit of our detection system. The width of the discharge at halfheight is 1.3 psec and the output falls below 0.2% of the peak value within 5 psec. Thymine solutions were prepared using Calbiochem A grade thymine in triply quartz-distilled water and concentrations were calculated using an extinction coefficient of 7.9 X 103 M-I cm-l a t X 264 nm and p H 7.4 Spectra of such solutions a;t p H 8 and p H 12 are shown in Figure 1. No transients are detectable at neutral pH in this water alone. Sodium hydroxide solutions were prepared fresh using Analar Reagent Grade NaOH and water prebubbled with Nz. p H adjustments were then made into prebubbled solutions by the addition of this freshly prepared material. Nitrogen and oxygen used in bubbling were Prepurified Grade from Canadian Anaesthetic Gases, Ltd. Various oxygen concentrations were obtained by bubbling O2-NZmixtures prior to photolysis and concentrations were calculated assuming the solubility of 0 2 in water as 1.3 X M at 25", and the per cent oxygen in the mixture. Nitrous oxide of similar quality was also obtained from this supplier. Tertiary buThe Journal of Physical Chemistry, Vol. 76,No. 4, 1079
x Figure 1. Ground-state absorption spectra of materials used in these experiments including the 10-2 M sodium hydroxide matrix, the 1 M MnSOa filter and the neutral and ionized forms of thymine, 7 x 10-5 M . Path lengths are 1 cm. Also indicated are the two tautomeric forms of the anion described by Wierzchowski, et a1.19
tanol and manganous sulfate were of reagent grade quality. All spectra were obtained through the point-by-point method with a reference wavelength using two monochromators and a Tektronix 556 dual beam oscilloscope. After passing through the cell the analyzing beam was split so that about 10% passed to a Bausch and Lomb High Intensity monochromator set to a fixed wavelength as reference. The main portion of the beam passed on through a preprism to a Spex monochromator. Detection by both systems was made using either an R136 or R196 photomultiplier tube (Hammamatsu TV Company). The incident analyzing light level at both wavelengths was sampled immediately before the flash and the absorptions of the species (6) (a) K. Berens and K. L. Wierschowski, Photochem. Photobiol., 9, 433 (1969); (b) W. Hauswirth and M. Daniels, ibid., 13, 157 (1971). (6) M. A. Herbert, J. C. LeBlanc, D. Weinblum, and H. E. Johns, ibid., 9, 33 (1969). (7) K. Schmidt and E. J. Hart, Advan. Chem. Ser., 81, 267 (1968). (8) J. W. Boyle, J, A . Ghormley, C. J. Hochanadel, and J. F. Riley, J.Phys. Chem., 73, 2886 (1959). (9) J. Rabani, W. A. Mulac, and M. J. Matheson, ibid., 6 4 , 5 3 (1965). (10) C. L. Greenstock, M. Ng, and J. W. Hunt, Advan. Chem. Ser., No. 81, 397 (1968). (11) C. L. Greenstock, J. W. Hunt, and M. Ng, Trans. Faraday Soc., 65, 3279 (1969). (12) D. W. Whillans, M. A. Herbert, J. W. Hunt, and H. E. Johns, Biochem. Biophys. Ras. Commun., 36, 912 (1969). (13) J. C. LeBlanc, M. Herbert, D. W. Whillans, W. B. Taylor, and H. E. Johns, unpublished results. (14) G. J. Fisher, J. C, LeBlanc, and H. E. Johns, Photochem. Photobhl., 7 , 767 (1967).
PHOTOREACTIONS IN AQUEOUS SOLUTIONSOF THYMINE, p H 12
l----Fielden E.M.ond Hari E.J. /‘a Trans. Faraday S0c.a 2975 (1967)
8
o’6
g
8
t
491
)
0‘4-
0.2
-
Figure 3. Quenching of the hydrated electron signal by (a) Oa in the absence of thymine, and (b) thymine in the absence of oxygen. For both cases the initial points represent poor exponentials, as explained in the text. 500
620
nm
x
Figure 2. Absorption spectrum of strongly absorbing transient produced in the flash photolysis of 10-2 M NaOH. Also shown is the spectrum of the hydrated electron obtained by Fielden, et al., by pulse radiolysis.16
were determined from the transient levels and these incident values. All absorptions were then normalized to the reference level to correct for variations in flash intensity. The analyzing beam itself was produced by pulsing an Osram XBO 150 W/1 xenon lamp to 15 times its continuous level from a current source for a 20-msec period surrounding the flash. This beam then passed three times through the 15-cm cell and to the monochromators. The noise level on the beam corresponds to an average absorbance of 0.0003 over a period of 100 psec. Rate constants for decays were obtained from oscillographs using an analogue to digital converter and a computer regression analysis for the exponential decays. Most points represent the result of several traces. The second-order plots were analyzed by hand.
Results and Discussion Species in Alkaline Water. When
M solutions of NaOH are flashed in our system, two very weak and one strongly absorbing species are observed. These transients all disappear when the exciting light is filtered by a 1 M MnS04 solution placed in a 0.5-cm concentric jacket around the cell. From Figure 1 it is clear that the transients are due to excitation of the NaOH. Figure 2 shows the spectrum of the strongly absorbing transient, which peaks a t -720 nm and which is completely quenched by N20. Also shown by the dashed line is a spectrum obtained by Fielden and Hart for the aqueous electron in the pulse radiolysis of water.15 It has a half-life of.70 psec or more in carefully degassed solutions, and its decay is nonexponential, as observed by others,lBand varies with flash
intensity. This species is efficiently quenched by 0 2 in a pseudo-first-order reaction, as shown in Figure 3a. The value for the bimolecular quenching rate of 1.6 X 1O1O M-l sec-* agrees well with the published value of 1.9 X 1O1O M-l sec-l.16 From this evidence the species has been identified as the aqueous electron. One of the weakly absorbing species whose yield is just above noise level is observed at wavelengths below 300 nm. This species is longer-lived than eaq- and not quenched by NzO, but is removed by the addition M tertiary butanol. On this basis it is the of hydroxyl radical, .OH, which is produced along with eaq- according to eq 1. A second weakly absorbing species is observed in the region 350-450 nm in solutions which have not been carefully deoxygenated. The species builds in over 50 psec or more at low 0 2 concentrations and is observed only when * OH is present. From this evidence it is the ozonide ion, Oa-, which is formed at this pH from interactions between O - ( - O H = O - + HfpK-11.9) and02.17 When M NaOH solutions are prepared containing small concentrations of thymine, the principal absorbing transient remains e,,-, but its rate of decay is increased by thymine as illustrated in Figure 3b where the exponential decay rate of e&,-, observed at 550 nm, is plotted against thymine concentration. A linear relationship is observed with a quenching rate constant of 3.0 X lo9 M-l sec-’. Since very few thymine molecules are excited under these conditions the reaction is between ea,- and the ground state thymine anion. The rate constant agrees well with that (15) E. M. Fielden and E. J. Hart, Trans. Faraday Soc., 63, 2975 (1967). (16) 8. Gordon, E. J. Hart, M. S. Matheson, J. Rabani, and J. K. Thomas, Discuss. Faraday SOC.,36, 193 (1963). (17) G . E. Adams, J. W. Boag, J. Currant, and B. D. Michael, in “Pulse Radiolysis.” M. Ebert, J. P. Keene, A. J. Swallow, and J. H. Baxendale, Ed., Academic Press, New York, N. Y . , 1965, Chapter 9.
The Journal of Physical Chemistry, Vol. 76, No. 4, 1978
D. W. WHILLANSAND H. E. JOHNS
492
1.c
P
9
4
I
Thymine pH 12 Short-lived species
2 P Q
9 0.:
g
c
I
500
400
h
600nm Ea*,,
