8186
J . Phys. Chem. 1990, 94, 8186-8192
calculated by a conventional cylindrical model (-1 0.4 ~ m ~ / m o l ) . ~ Values for AV-l* were estimated from the values of AV2*and AVZf - AV-,*given in Table 1. These results are also listed in Table I, together with relevant activation volumes. .AV-,* is equal to At',*, within experimental error, which suggests that the step associated with k-, may also be fully diffusion-controlled. This absence of a volume change upon complex formation implies that an encounter complex is involved in the reaction mechanism. In conclusion, the present high-pressure study provides quantitative information regarding the pressure-induced viscosity dependence of the nature of the primary process of benzophenone photoreduction. The origin of the curvature in the plots of In k , against pressure may arise due to the effects of diffusion on the
activation-controlled hydrogen-abstraction process, where these effects increase gradually with increasing pressure. Also, the value of AV21 is found to be independent of solvent polarity since AVz* - A Y 1 *correlates linearly with AV,*(300 MPa), irrespective of solvent polarity. This result is in agreement with our previous conclusion that the triplet is radical like in r e a ~ t i v i t y . ~
Acknowledgment. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education of Japan (No. 62540331). Registry No. Benzophenone, 1 19-61-9; 5-methyl-3-heptano1, 1872065-5; 2-octanol. 123-96-6; 2-methyl-I-propanol,78-83-1; 2-propano1, 67-63-0.
Direct Evidence for the Formation of Thymine Radical Cations from the Reaction of SO,- with Thymine Derivatives: A Pulse Radiolysis Study with Optical and Conductance Detection David J. Deeble,* Man Nien Schuchmann, Steen Steenken,* and Clemens von Sonntag* Max-Planck-Institut fur Strahlenchemie, Stiftstrasse 34-36, 0-4330 Mulheim a.d. Ruhr, Germany (Received: April 10, 1990)
A number of N( 1)-substituted thymine derivatives have been oxidized in aqueous solution using the one-electron oxidant SO,'-.The rate constants for this reaction are in the range 1 X IO9 mol-l dm3 s-I (5'-thymidylic acid) to 5 X lo9 mo1-I dm3 s-I (1-methylthymine). It is proposed that in all cases a radical cation is the initial transient observed. The 1,3-dimethylthymine radical cation decays by a first-order process (tIl2 2 W S ) with the production of a proton and can be followed by both absorbance and conductance measurements. This process is made up of water addition at C(6) and deprotonation from the C(5) methyl group. In alkaline solution the rate constant for the decay of the radical cation is increased, with OH- addition at C(6) being preferred to deprotonation from the C(5) methyl group. When N(3) is unsubstituted, deprotonation of the radical cation can occur to give the N(3)-centered radical. The pK, values for thymidine and 1-methylthymineradical cations are 3.6 and 3.8, respectively, Le., more than 5 orders of magnitude lower than those of their parent compounds. These radical cations also decay by a first-order process, again involving both water addition at C(6) and deprotonation from the C(5)-methyl group ( t l 1 2= 0.9 and 2.4 CIS, respectively, for the radical cations of thymidine and 1-methylthymine). Around neutral pH essentially only the N(3)-centered radicals are present and these decay bimolecularly (2k = IO9 mol-] dm3 s-I). The first-order decay of the radical cations is unaffected by the presence of oxygen (1.3 X mol dm-3). On the other hand the carbon-centered radicals produced in this decay react rapidly (close to diffusion controlled) with oxygen. The N(3)-centered radical reacts with oxygen with a rate constant of only 3.6 X lo7 mol-' dm3 s-l.
-
Introduction
In living systems, ionizing radiation causes a number of deleterious effects ranging from reproductive cell death to mutagenesis and transformation. These effects are mainly due to lesions induced in cellular DNA, which is believed to be the prime target for the action of ionizing radiation. Ionization can either occur within the DNA itself, the direct effect, or in its near vicinity, producing reactive species (e.g., water-derived radicals) which then attack the DNA, the indirect effect. The reactions of the water-derived radicals with DNA and its components have been extensively studied in dilute aqueous so1utions.l In contrast, our knowledge concerning the direct effect is very limited, chiefly for reasons of a practical nature. Whereas experimental conditions can easily be arranged so that essentially only the indirect effect occurs (e.g., by irradiating dilute aqueous solutions), it is much more difficult to design an experiment where only the direct effect operates. Direct ionization of DNA leads to the formation of electrons and DNA radical cations. Radiolytically produced nucleobase radical cations have been studied in a freon matrix.2 In aqueous solution they can be conveniently ( I ) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor and
Francis: London, 1987. ( 2 ) Rhodes, C. J.; Podmore, I. D.; Symons, M. C. R. J . Chem. Res. Synop. 1988, 120.
0022-3654/90/2094-8 I86$02.50/0
generated from their parent nucleobases by using either phoor strongly oxidizing species such as excited quitoi~nization)-~ nones (e& menadione),'&1s Tl2+,I6sl7Br98%) of these preparations was verified by HPLC. Pulse radiolysis was carried out using a 2.8-MeV Van de Graaff electron generator delivering 0.4-ps electron pulses of 3-10 Gy. The pulse radiolysis setup with the optical and conductance detection techniques as well as the data processing procedures have
1
-lo4 x
-Time
Pulse
-
Figure 2. Conductance change following an electron pulse of ca. 8 Gy in an argon-saturated solution of 1,3-dimethylthymine (5 X IO4 mol mol dm-') and tert-butyl alcohol ( 5 dm-)) containing K2S208(5 X X mol dm-)) at pH 4.2.
TABLE I: Rate Constants for the Reactions of SO,'- with Some Pyrimidines pyrimidine 1,3-dimethyluracil 1,3,6-trimethyluracil 1-methylthymine 1,3-dimethylthymine 1,3,6-trimethylthymine thymidine 3-methylthymidine 5'-thymidylic acid
k/dm3 mol-' s-I 5.5 x 109' 3.5 x 109 5.0 x 109 4.6 x 109 4.1 x 109 2.1 x 109 3.5 x 109 1.0 x 109
From ref 26.
been d e s ~ r i b e d . ~ ' . ~ ~ Dosimetry in pulse conductance measurements was made relative to solutions in which the pyrimidines were replaced by 0.05 mol dm-3 methanol, taking a radiolytic yield for proton formation of 6.2 X lo-' mol J-I. Although in this system the this reaction is relatively methanol radicals react with S2082-,39 slow and does not interfere on the time scale of interest ( 1 2 0 ps). Solutions of the pyrimidine derivatives were prepared in water purified with a Millipore Milli-Q system. The solutions typically to lW2 mol dmJ of SzO& anions and 0.05-0.5 contained 5 X mol dm-3 tert-butyl alcohol as O H radical scavenger and were saturated with argon. Most of the irradiations were carried out at 20 OC using a thermostat; some were at room temperature (18 f 2 "C).
Results and Discussion The Rate Constants for the Reaction of SO4'- with Thymine Derivatives. The SO4'- radicals generated according to reactions (38) Steenken, S.; Buschek, J.; McClelland, R. A. J. Am. Chem. Soc. 1986, 108, 2808.
(39) Schuchmann, H.-P.; von Sonntag, C. Radiat. Phys. Chem. 1988.32, 149.
8188 The Journal of Physical Chemistry, Vol. 94, No. 21, 1990 2 and 3 have a strong absorbance with the maximum at around 450 nm.40341 The reaction of SO4'- with various pyrimidines has been followed by using pulse radiolysis with optical detection. Bimolecular rate constants were determined from the slopes of the linear plots of the observed pseudo-first-order rate constants against the pyrimidine concentrations (cf. Figure 1). The values range between 1 X lo9 and 5 X lo9 dm3 mol-' s-I (Table I ) and are close to that reported for 1,3-dimethyl~racil.~~ Our thymidine value differs considerably from that determined by competition kinetics ( k I2 X lo8 dm3 mol-').30 The method used here directly follows the reaction and is therefore probably more reliable than the competition technique which could contain unforeseen complications. Conductance measurements (see below) confirm our values. 1,3-Dimethylthymine (I,3-Me2T)and 1,3,6-Trimethylthymine (1,3,6-Me3T).In the reaction of SO4*-with olefins, SO,'--adduct radicals have been ~ b s e r v e d . ~Thus, ~ . ~ ~it is reasonable to assume that an adduct is formed initially on the reaction of SO4'- with pyrimidines ( e g , reaction 6). Pulse conductance measurements
Deeble et al.
* S I . 1
2 I
300
I
LOO -A)i/nm
I
500
600
d
Figure 3. Spectra of transients (normalized to 1 Gy) from the reaction of 1,3-dimethyIthyminewith SO4'- at pH 4.4. Argon-saturatedsolution of 1,3-Me2T(5 X IOd mol d d ) containing K2S208 (0.01 mol dm-') and tert-butyl alcohol (0.05 mol d ~ n - ~(A) ) . 1.5 p s and (X) 15 p s after the pulse. Inset: Spectrum of an 0,-saturated solution of 1,3-Me2T,composition as above, measured 15 p s after the pulse.
This can be interpreted in two ways, either the SO4'- adduct formed in reaction 6 is relatively long lived or it dissociates rapidly to give the 1,3-Me2T radical cation and an S042-ion (reaction 7). It is difficult to distinguish between these two possibilities because the difference in the conductance of H+ and the SO4'adduct on the one hand and (the slightly higher) conductance of H+,S042-,and the I,3-Me2T radical cation on the other is small compared to the total conductance. The occurrence of a further increase in conductance in a rapid second step (reactions 8 and 9) diminishes any possibility of achieving such resolution to the extent of infeasibility. Nevertheless, there is substantial evidence favoring a fast elimination with the formation of a relatively long-lived radical cation and this will now be presented. In Figure 3 the transient absorption spectra at 1.6 and 15 p s after the pulse are given. At the 1,3-Me2T concentration used reaction 6 is completed in < I ps. Hence, the initial spectrum (A in Figure 3) is mainly (=60%) due to either the SO4'- adduct or the radical cation, the remaining contribution being due to the presence of some of the product ( X in Figure 3) formed even at these short times in the rapid first-order decay of the initial transient. The rate of this first-order decay was found to be independent of the 1 ,3-Me2Tconcentration and occurs with a rate
constant of 3.5 X IO5 s-I at pH 4-5. This value is in good agreement with that observed when conductance changes are monitored ( k = 3.7 X lo5 s-l, see Figure 2 ) . On saturating the solution with oxygen ([O,]= 1.3 X mol d ~ n - the ~ ) initial spectrum at pH 4.1 was unaltered and decayed with the same rate constant as in the absence of oxygen. However, the spectrum of the resulting transient was now different (inset in Figure 3). An SO4*-adduct where the radical site would be at either C(6) or C(5) should react with oxygen with a rate constant of about 2 X IO9 dm3 mol-' SKI as has been found for other pyrimidineadduct radicals.44 If this were the case the decay of the initial transient should be enhanced by the presence of oxygen. On the other hand, radical cations are known to react with oxygen at best only slowly (cf. refs 45 and 46). Experiments on 1,3,6-Me3Tconclusively showed that reaction with SO4'- produced a long-lived radical cation (t1/2 = 20 ps). The greater stability of this radical cation enabled precise comparisons to be made of the initial conductance change of this system with systems which are known to produce an adduct radical or a radical cation on reaction with SO4*-. Adduct radicals,
