J . Phys. Chem. 1991, 95, 9340-9346
9340
these two compounds differ primarily by the number of a C-H bonds. The similar kinetic behavior of C4H80 and C 6 H I 2 0is in agreement with the assumption that their a C-H bond strengths are equal, as discussed above. The intermediate reactivity of CSHloOcertainly agrees with a calculated a C-H bond strength between those of the dioxanes and those of C4H80and C6HI20. The behavior of C3H60, however, is somewhat surprising. The calculated value of its a C-H bond strength, 2, is comparable to the corresponding one in oxolane 5. Yet, its reactivity is intermediate. Also, its activation energy is about the same as for p-C4H802and m-C4H8O2,with its higher reactivity apparently arising from a higher preexponential factor. We note that C3H60 has a relatively high charge density on the a-carbon atoms and
the low calculated bond strength results from the lower degree of puckering in the oxetanyl radical. Therefore, one must exercise some caution when attempting to use the results of gas-phase theoretical computations to understand the reactivity of molecules in solution, especially since the effects of solvent as well as other ionic effects are not included in the computations.
Acknowledgment. This research was supported by the Office of Basic Energy Sciences of the US.Department of Energy. Dr. S. A. Kafafi acknowledges the financial support of the A. Mellon Foundation. Registry No. 1, 287-23-0; 2, 503-30-0; 4, 287-92-3; 5, 109-99-9; 7, 110-82-7; 8, 142-68-7; 12, 505-22-6; 14, 123-91-1; 15, 110-88-3; 16, 291-64-5; 17, 592-90-5; Sod'-, 12143-45-2.
Pattern of OH Radical Reaction with N6,N6,9-Trimethyladenine. Dehydroxylation and Ring Opening of Isomeric OH Adducts A. J. S.C. Vieira' and S. Steenken*** Max-Planck-Institut fur Strahlenchemie, 0-4330 Mulheim, Germany, and Instituto Superior TEcnico, P- 1096 Lisboa, Portugal (Received: April 4, 1991)
The OH radical reacts with M,M,9-trimethyladenine (A) in aqueous solution by addition to carbons 4 and 8 of the purine system ( k = 8.4 X lo9 M-I s-I). The resulting radicals A40H' and ASOH' undergo elimination of OH- (identified by conductance; kel= 2 X IO6 s-l) and ring opening ( k = 2.3 X lo5 s-l), respectively. The two types of reaction have different activation parameters. The (heterolytic) dehydroxylation reaction of A40H' is inhibited by H+and by OH-. The radical cation (the yield per 'OH is 50%) formed by elimination of OH- from A40H' is oxidizing (with respect to N,N,N',N'tetramethyl-p-phenylenediamine);in contrast, A80H' or its ring-opened product is reducing (toward tetranitromethane or viologens). On one-electron oxidation, ASOH' is converted into 8-hydroxy-Nd,Nd,9-trimethyladenine,which was measured by HPLC with optical and electrochemical detection.
Introduction Of the constituents of DNA, the purines and the pyrimidines are the most sensitive to the destructive effects of ionizing radiation in aqueous s ~ l u t i o n . ~It has therefore been the aim of radiation chemists and biologists to understand in detail the mechanisms by which ea;, 'H, and 'OH, the radical products of the radiolysis of water, interact with these heterocycles. Due to the simple structure of pyrimidines as compared to purines, progress in understanding the radical chemistry has been more pronounced with the formere7 than with the A (1) lnstituto Superior Tecnico. (2) Max-Planck-lnstitut. (3) For reviews, see, e.g.: (a) Scholes, G.In Photochemistry and Photobiology of Nucleic Acids; Wang, S.Y . , Ed.; Academic Press: New York, 1976; Vol. 1 , p 521. (b) HOttermann, J., Kbhnlein, W., Teoule, R., Bertinchamp, A. J., Eds. Eflects offonizing Radiation on DNA; Springer: Berlin, 1978. (c) Myers, L. S. In Free Radicals in Biology; Pryor, W. A., Ed.; Academic Press: New York. 198O;.Vol. 4, p 95. (d) Cadet, J.; Berger, M. Int. J . Radial. Biol. 1985,47, 127. (e) von Sonntag, C.; Schuchmann, H.-P. Int. J . Radiat. Biol. 1986, 49, I . (4) Fujita, S.; Steenken, S.J . Am. Chem. SOC.1981, 103, 2540. (5) Hazra, D. K.; Steenken, S.J . Am. Chem. SOC.1983, 105, 4380. (6) Jovanovic, S.V.; Simic, M. G. J . Am. Chem. Soc. 1986, 108, 5968. (7) Schuchmann, M. N.; Steenken, S.;Wroblewski, J.; von Sonntag, C. Int. J . Radiat. Biol. 1984, 46, 225. (8) (a) ONeill, P. Radiat. Res. 1983, 96, 198. (b) ONeill. P. In LVe Chemistry Reports Supplement 2, Oxidative h m a g e and Related Enzymes; Rotilio, G . , Bannister, J. V., Eds.; Harwood Academic Publishers: London, 1984; p 337. (c) ONeill, P.;Chapman, P. W. Int. J . Radial. Biol. 1985, 47, 71. (d) ONeill, P.; Chapman, P. W.; Papworth, D. G. Life Chem. Rep. 1985, 3, 62. (9) (a) Vieira. A. J . S.C.; Steenken, S.J . Am. Chem. SOC.1987, 109, 7441; (b) J . Phys. Chem. 1987, 91,4138. (c) Candeias, L. P.; Steenken, S. NATO AS1 Ser., Ser. H 1991, 54, 265. (10) Vieira, A. J . S.C.; Steenken, S.J . Am. Chem. SOC.1990, 112, 6986.
feature that complicates the chemistry of the purine radicals is that, in contrast to the pyrimidines, the radicals produced on reaction with 'OH undergo unimolecular transformation react i o n ~ In . ~the~case ~ ~of ~adeninederivatives ~ (=A) it has recently been shown that there are at least two different radical isomers involved, which have been identified as the radicals formed by addition of 'OH at C48a and at C8,"J0J1 abbreviated as A40H' and A80H', respectively. A80H' has been suggested to ring pen^^,^,^^*^^,^^ and A40H' to undergo dehydroxylation, which, if there is a t least one mobile proton at (the exocyclic) N6, is equivalent to a dehydration reaction, due to deprotonation from the (incipient) radical cation. However, in the case of fully alkylated adenines such as M,N6,9-trimethyladenine,dehydration reactions of OH adducts should show up as elimination of OH- (which can be monitored by conductance). On this basis, trimethyladenine is expected to be a suitable model to test the hypothesis that one of the transformation reactions is a dehydroxylation, and to further study the substituent effect on the transformation reactions of O H adducts of purines described p r e v i o ~ s l y . ~ ~
Experimental Section N6,M,9-Trimethyladenine (abbreviated as A) was prepared by methylation of N6,M-dimethyladenine (from Fluka) with CH31 (1 1) Steenken, S.Chem. Rev. 1989,89, 503. (1 2) Scholes, G.; Shaw, P.; Willson. R. L.; Ebert, M. In Pulse Radiolysis; Ebert, M., Keene, J. P., Swallow, A. J., Baxendale, J. H., Eds.; Academic Press: London, 1965; p 151. Michaels, H. B.; Hunt, J. W. Radiat. Res. 1973, 56, 57. Wold, E.; Brustad, T. Int. J. Radiat. Biol. 1973, 24, 153. Fel, N. S.; Zaozerskaya, L. A. Zfi-Mitt. 1981, 436, 273. (13) van Hemmen. J. J. Int. J . Radiat. Biol. 1975, 27, 403.
0022-3654 19112095-9340%02.50/0 0 , - 1991 American Chemical Societv I
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The Journal of Physical Chemistry, Vol. 95, NO.23, 1991 9341
Pattern of O H Radical Reaction 10000
Figure 1. Time-resolved absorption spectra measured after reaction of OH' with 1 mM trimethyladenine at pH 6.7 and 20 OC. Squares, 0.9 ps; asterisks, 10 j t s after the pulse. z values are based on G(adducts) = 6.0. Insets: changes in the OD or conductance following the formation of the adducts.
and it was separated by column chromatography and purified by TABLE I: Rate Constants for Reaction of OH' and SO,'- with A and recrystallization from n-hexane until only one spot on TLC was for Transformation of A40H' and A80H' vi~ib1e.l~Analytical grade rerr-butyl alcohol, K2S208: K,Fe(CNh, EA, a', (Merck), and N,N,N',N'-tetramethyl-p-phenylenediamine2HCl k, s-I kcal cal log (Fluka) were used as received. The aqueous solutions (water radical/reaction (20 "C) mol-' a (mol K)-l a A, s-I purified with a Millipore-Milli-Q system) typically contained 0.1-1 A t OH' 8.4 x 109b'~ mM purine and they were saturated with N 2 0 in order to convert 4.0 x 109b.d A +S O:e, into 'OH. For experiments aimed at scavenging radicals with A40H'/OH2.2 X 8.9 -1.5 12.9 02,the solutions were saturated with a 4:l mixture of N 2 0 and elimination 1.8 x 106' 02.The pH of the solutions was adjusted with HCIO,, NaOH, 2.1 x 1068 A80H'/ring opening 2.3 X losh 4.2 -2 1 8.5 or phosphate ( 1 mM). pK,(AH+ e The 3-MeV van de Graaff accelerator delivered electron pulses A + H+) = 4.04' of 0.1-0.4-ps duration with doses such that 1-2 pM radicals were produced. The optical and conductance (from an ac or a dc The activation parameters were determined by optical measureinstrument) signals were digitized by a Biomation 8100 transient ments at pH 6-8. Concentration of A = 0.4-1 mM. bThe units are recorder interfaced with a VAX 11/38 computer via a PDP 11/10. M-I s-l. CFrom the buildup of OD at 320 and 480 nm. The error limits are *lo%. [Substrate] = 0.02-0.2 mM, pH -7,20 OC. dFrom With optical detection, dosimetry was performed by using N 2 0 the buildup of OD at -350 nm, pH -6. eFrom the OD decay at saturated IO mM KSCN solutions for which G(0H) = 6.0 and 410-430 nm, pH -7. fFrom the OD buildup at 5 7 0 6 2 0 nm, pH -7. C((SCN)~-)at 480 nm = 7600 M-' cm-I.l5 For experiments with #From conductance increase at pH 9.5-10.5. "ram OD buildup at conductance detection two dosimetry systems were used: (a) a -370 nm. 'Determined by monitoring the OD at 275 nm as a funcN,O-saturated 0.2 mM solution of dimethyl sulfoxideL6at pH 4.3 tion of pH. and 9.3 for which G(H+) (at pH 4.3) = G(-OH-) (at pH 9.3) = 6.0" (assuming 314, 174, and 35 cm2 Q-I mol-' for the specific conductance of H+, OH-, and the sulfinate ion, respectively) and M,N6,9-trimethyladenine and its 8-hydroxy derivative. (b) a N20-saturatedsolution at pH 1 1.3 that contained 0.1 mM Results and Discussion phenol, for which G(+OH-) was found to be 6.0 f 0.2 compared to solution (a) under both acid and basic conditions, using a specific (1) Formation of OH Adducts and Their Transformation Reconductance for the phenolate ion of 35 cm2 f2-l mol-I. With the actions. (a) Optical Detection. In Figure 1 is presented the optical measurements the solutions were thermostated at 20 f spectrum of the transients formed initially on pulse irradiation 0.1 "C with cells that are an integral part of a heat exchanger. of an N,O-saturated aqueous solutions of 1 mM Nd,N6,9-triWith the 6oCo y-radiolysis experiments, the dose rate was 1 methyladenine (=A) (spectrum "0.9 ps"). After the pulse, the krad/min. Product analysis was performed with HPLC using a rate of increase of optical density (OD; monitored at 320 and 480 4.6 X 125 mm Nucle0sil-5-C~~ column. The eluent was 70:30 nm), koW, was found to be proportional to the concentration of (v/v) water/methanol containing 2 mM KH2P04 and 20 mM A (in the range 0.02-0.2 mM), and from this linear dependence NaCIO,. The flow rate was 0.8 mL/min. Optical (at 254 nm the rate constant for reaction of 'OH with A (at pH 7) is obtained or with diode array) and electrochemical detection (voltage 0.7 as 8.4 X lo9 M-' s-l (see Table I). This value is somewhat larger V) were used. Retention times were 3.6 min for resorcinol (used than that (6.1 X lO9)I8 for reaction of (the electrophilic) 'OH with as internal standard) and I5 and 1 1 min respectively for adenine. This increase in reactivity probably reflects the larger electron density that results from methylation of adenine. The spectrum 0.9 ps of the initially formed transients shows (14) Itaya, T.; Matsumoto, H.; Ogawa, K. Chem. Pharm. Bull. 1980,28, maxima at -350, -425, and 2520 nm. This spectrum undergoes 1920. time-dependent changes to yield the spectrum "IO ps". The insets (IS) Schulcr, R. H.; Hartzell, A. L.;Bchar, B. J . Phys. Chem. 1981,85, 192. a, b, c, and e in Figure 1 show the OD changes by which the (16) Veltwisch, D.;Janata, E.;Asmus, K.-D. J . Chem. Soc.,Perkin Trans. N
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2 1980, 146. (17) Steenken, S.;Buschek, J.; McClelland, R.A. J. Am. Chem. Soc. 1986, 108, 2808.
(18) Buxton, G. V.;Greenstock, C. L.;Helman, W.P.; Ross,A. B.J . Phys. Chem. Re/. Data 1988, 17, 513.
Vieira and Steenken
9342 The Journal of Physical Chemistry, Vol. 95, No. 23, 1991
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Figure 2. Dependence on pH of the rate constants at 20 O C for transformation of A40H' and A80H'. [Trimethyladenine] = 0.4 mM. Asterisks, decay at 420 nm (=dehydroxylation of A40H'); open squares, buildup at 370 nm (=ring opening of A80H'). No buildup at 370 nm is seen below pH -4. The full squares refer to a buildup at 340 nm. initially observed absorption spectrum is transformed into the resulting spectrum. At -430 nm the optical density due to the initially formed transient(s) decreases rapidly in an exponential way (see inset a), whereas at -370 and >530 nm the OD increases, again exponentially (see insets b, c, and e). The rates of buildup of OD in these two wavelength regions are, however, different (compare b or c with e). The rates of these processes were found to be independent of the concentration of (a) the substrate (in the range 0.4-2 mM) and (b) the radicals initially produced (1-10 pM), from which it is concluded that the reactions are unimolecular. The rates are also independent of pH between pH -6 and 10. It is interesting that the rate of increase of OD measured at >530 nm (k = 1.8 X IO6 s-l) is equal within experimental error (&IO%) with that of the decrease at -430 nm (k = 2.2 X IO6 s-I). The rate of increase of OD at -370 nm is, however, considerably lower, i.e., by almost a factor of 10 (see Table I and insets in Figure 1). In order to characterize the transformation reactions further, their activation parameters were determined by measuring the rate constants as a function of temperature in the range 0-30 OC in steps of 5 deg, for the slow buildup at 350-370 nm and the fast decrease at 430 nm. From the Arrhenius plots of the rates (correlation coefficients 20.99) the parameters listed in Table I were obtained: the slow reaction (buildup at -370 nm) is characterized by a low activation energy (-4 kcal mol-I) and by a very negative activation entropy (-21 eu). In contrast to this are the parameters for the (fast) decrease at -430 nm: both the activation energy (-9 kcal mol-I) and the activation entropy (-( 1-2) eu) are considerably larger than those for the slow reaction. Comparing the two reactions, it is thus seen that, with the fast reaction, the rate-retarding effect of the higher activation enthalpy is overcompensated by the rate-enhancing effect of the larger activation entropy. The slow and the fast reactions differ also with respect to their dependence on pH: in Figure 2 it is shown that the rate of the fast reaction decreases drastically with decreasing pH below pH 6. The rate decreases also between pH 11 and 12. In contrast, the rate of the slow reaction remains essentially constant between pH 4 and 13. Below pH -4 there is a change in the absorption spectrum of the radical(s): the species formed in the unimolecular buildup now has a maximum at -340 nm. (b) Conductance Detection. The reaction between 'OH and A results in a change of conductance: As shown in inset d of Figure 1, on a fast time scale an increase of conductance is seen when the reaction is carried out at pH 9-1 1, whereas a decrease of conductance is observed at pH 4-6 (not shown). The rate of this fast conductance change, as measured at pH 10 ( k = 2.1 X lo6 s-l), is essentially equal with that of the fast decrease of OD a t 430 nm or of the increase at >530 nm observed optically (see Figure 1 and Table I). This indicates that with the two methods the same reaction is being monitored. The reversal of the polarity of the conductance signal on going from basic (positive signal) to acid medium (negative signal) proves that one of the ions produced is OH-. Le., at pH
