1027
J. Phys. Chem. 1981, 85, 1027-1031 Residual Spectra From 0311OTorr)-$H, (O5Torr)-Air i700Torr) Mixture
( A ) O 3 ( Z T o r r ) - C Z H q ( I T o r r ) in A l r
At
2(
3 min.
CHZO(.31)
+--
( A l l =Omin -1760
1
(C) = 2 0 m i n
( E ) Residual Spectrum ( x 2 )
I -1000
2943
I
A
3405 i 2000
I coo
1500
l/X.
cml
I
Figure 7. Effect of sample aging on the residual spectra in the frequency region of 600-2000 cm-’ from a mixture initially containing O3 (1 torr) and C2H, (0.5 torr) in 700 torr of air: (A) recorded about 2 min after mixing the reactants; (6)and (C) 10 and 20 min aged samples.
I
3500
3000 I/X
( cm“)
Figure 8. Absorbance spectra in the frequency region of 2700-3700 cm-‘ from a mixture initially containing O3(2torr) and C2H4(1 torr) in 700 torr of air: (A) about 3 min reaction time; (B) residual spectrum of (A). Values In parentheses are pressures In torr.
transformation at high concentrations rather than decaying heterogeneously to formic anhydride as observed a t low concentrations. Formation of aerosols and liquid products in the 03-olefin reactions in the torr region is well documented.13 In fact, light scattering by aerosols could be readily observed with a He-Ne laser under these conditions. An FT IR observation of aerosols generated in situ by gas-phase chemical reactions has been reported pre-
viou~ly.’~Thus, it appears that compound X is a major aerosol precursor a t least in the 03-C2H4reaction a t high reactant concentrations. It is also conceivable that because of the gas-to-aerosol conversion this species escaped detection in the matrix-IR and mass spectrometric studies reported e a r l i e ~ ~ J
(13) P. A. Leighton, “Photochemistry of Air Pollution”, Academic Press, New York, 1961.
(14) H. Niki, P. D. Maker, C. M. Savage, and L. P. Breitenbach, J. Phys. Chem., 84, 14 (1980).
ESR Study of DNA Base Cation Radicals Produced by Attack of Oxidizing Radicals Michael
D. Sevllla,
Darbha Suryanarayana, and Kim M. Morehouse
Department of Chemistty, Oakland University, Rochester, Michigan 48063 (Received: September 30, 1980; In Final Form: December 23, 1980)
ESR investigations of y-irradiated aqueous glasses (8 M NaC104,12 M LiC1) containing DNA bases and analogues show that oxidizing species produced in the glasses (hydroxyl radicals and ClJ at 77 K attack a number of the bases and their analogues after warming to produce *-cation radicals. Compounds which produce T cations include thymine, uracil, 3-methyluracil, 6-methyluracil, orotic acid, isoorotic acid, guanine, and adenine. Compounds which are not found to produce T cations are 1-methylthymine, thymidine, and 1-methyluracil. The overall reaction for formation of the A cations is as follows: base + OH. (C12-) OH- (2Cl-) + base cation. A pH and substituent dependence is also noted for the pyrimidines. At pHs where the nitrogen at position 1 is protonated, *-cation radicals are not formed. In addition, when there are substituents at position 1, R cations are not found. Analysis of the ESR spectra of the DNA base ?r cations for hyperfine splittings and g values were performed by aid of computer simulations. The analyses are in agreement with values found for several DNA base cations produce by photolysis and by direct y irradiation.
-
Introduction There have been a number of reports that oxidizing .OH, and clz- may produce cations radicals such as so4-, of DNA bases and anal~gues.l-~The production of py-
rimidine a cations4by C1, reaction was first suggested by Ward and Kuo.’ In later work,2 to explain the results of product analysis of irradiated chloride containing solution
(1) J. F. Ward and I. Kuo, Int. J . Radiat. Bid. Relat. Stud. Phys., Chem. Med., 15, 293 (1969). (2) G. A. Infante, P. Jirathans, J. H. Fendler, and E. J. Fendler, J. Chem. Soc., Faraday Trans 1, 70, 1162 (1974); 70, 1586 (1974).
(3) M. B a n d and W. Fessenden, Radiat. Res., 75, 497 (1978). (4) The term “T cation” is used here to mean a radical produced by loss of an electron from the r-electron system and does not refer to the total charge on the molecule.
0022-3654/81/2085-1027$01.25/0
0 1981 American Chemical Society
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The Journal of Physical Chemistry, Vol. 85,
Figure 1. ESR spectrum of thymine in 8 M NaCi04/D20,pD E 11, y-irradiated at 77 K. The three markers in each spectrum are separated by 13.1 G. The central marker Is at g = 2.0056. (A) recorded at 100 K. (B) After warming to 170 K; recorded at 145 K. (C) The spectrum in 6 mlnus the background signal, showing the thymine a-cation radical.
of thymine, Fendler et al. also suggested that Clz- would react as in reaction 1. This reaction was only found to
Ciz-
i-
’& H3
O
Ht
+
2CI-
+
Ai
H0 -JycH3
Sevilla et ai.
No. 8, 1981
(1)
HI
occur at low pHs where Clz- formation is favored. Bansal and Fessenden suggested that analogous reactions occurred in their study of the reaction of SO4- and Clz- with uracil by ESR, pulse radiolysis, and conductometric technique^.^ They further suggested that OH. attack at pH 11may also result in the a cation of uracil and thymine-not ring opening as previously reporteda6 In our previous work we have reported the production of several DNA base a cations by photoionization.6 In this work we report the production of over 10 DNA base and analogue a cations by attack of oxidizing species produced by the radiolysis of aqueous glasses. The dependence of the production of the pyrimidine a cation on pH and substituents is reported.
Experimental Section The DNA bases and substituted bases were obtained from Sigma Chemical Co. or Aldrich Chemical Co., of the highest purity available, and were used without further purification. The LiCl used was the ultrapure grade obtained from the Alpha Chemical Co. Two glasses were employed in this investigation, 8 M NaC104 (DzO)and 12 M LiCl (DzO). DNA, bases M) were dissolved in the solutions, and the deoxygenated solutions were frozen and y-irradiated at 77 K to a dose of 0.05 Mrd for 8 M NaC104 glasses or 0.1 Mrd for 12 M LiCl glasses. The pD was adjusted between neutral and basic conditions. Generally pD near 11gave the best ESR signal, for the DNA base a cations in both glasses. In the 8 M NaC104 glass the y irradiation produces electrons as well as 0- (OD).7 The electrons react with the Clod- to (5) P. Neta, Radiat. Res., 49, 1 (1972). (6) (a) M. D. Sevilla, “Excited States in Organic Chemistry and
Biochemistry”, B. Pullman and N. Goldblum, Eds., Reidel, Dordrecht, Holland, 1977, pp 15-25; (b) M. D. Sevilla, J. Phys. Chem., 75, 626 (1971); (c) M. D. Sevilla, C. Van Paemel, and C. Nichols, ibid.,76, 3571 (1972); (d) M. D. Sevilla, C. Van Paemel, and G. Zorman, ibid.,76,3577 (1972); (e) M. D. Sevilla and P. A. Mohan, Int. J. Radiat. Biol.Relat. Stud. Phys., Chem. Med., 25, 635 (1974); (f) M. D. Sevilla, J. Phys. Chem., 80,1898 (1976); (9) M. D. Sevilla, J. B. D’Arcy, K. M. Morehouse, and M. L. Englehardt, Photochem. Photobiol., 29, 37 (1979).
Flgure 2. (A) ESR spectrum of 12 M LiCI/D20, pD 11, y-irradiated at 77 K and recorded at 100 K after photobleaching. (B) ESR spectrum of thymine in 12 M LiCiID20, pD 11, after y-irradiation at 77 K, showing the thymine anion doublet, g = 2.0036, recorded at 100 K. (C) Sample same as in B, but contains M K,Fe(CN)B,recorded at 100 K, showing the decrease of the thymine anion.
produce additional 0-and C10,. Even at the low doses employed a background signal in the NaC10, glasses is produced upon annealing. In 12 M LiCl two electron scavengers were employed, Fe(CN):M) and NzO (saturated). DNA base radicals were produced upon annealing glasses to temperatures where the oxidizing species produced by y irradiation of the glasses became mobile (ca. 165 K). Other experimental details are reported in previous publications.6
Results and Discussion DNA Base a Cations i n y-Irradiated 8 M NaC104 The y irradiation of 8 M NaC104 (DzO) containing thymine results in the spectrum found in Figure 1A. The signal due to SO- predominates in the spectrum and shows characteristic anisotropic g values (g = 2.002, g, = 2.053).7 Upon warming the 0- presumably deuterates to form OD. and reacts with the DNA base thymine, producing the spectrum in Figure 1B. Figure 1C shows the spectrum due to the DNA base after subtraction of the matrix background signal which is also produced on warming. Figure 1C is the characteristic spectrum of the thymine a cation.6b>fAnalysis gives an isotropic 20-G coupling due to the three methyl protons and an anisotropic nitrogen coupling ( A = 1 2 G, A L N = 0) due to the nitrogen at position 1. bhese values are the same as those found for the thymine a cation produced by photoionization of thymine in 8 M NaC10, and reported in earlier work.6f Guanine and cytosine were also investigated in 8 M NaC104. Spectra produced on warming had large background signals. However, after subtraction the a cations of guanine and cytosine could be recognized from comparison to spectra produced in 12 M LiCl (see later) which were free from the background signal. Results in the NaC104 glass suggest that OD. is able to oxidize the DNA bases studied and produce their a cations. The use of the NaC104 glass was limited because of the large matrix background signal, and much clearer results were found in 12 M LiCl glasses. DNA Base a Cations in y-Irradiated 12 M LiC1. y irradiation of 8-15 M LiCl glasses has been previously reported to produce e-, CI,, OH., and H-.’-lo In our system (7) L. Kevan, Chem. Biol. Action Radiat., 13, 57-117 (1969). (8) A. Plonka and T. S. Kowalski, Bull. Acad. Pol. Sci., Ser. Sci. Chim., 25, 885 (1977). (9) E. R. Andrew, H. J. Gale, and W. Vennart, J.Magn. Reson., 33, 289 (1979).
ESR Study of DNA Base Cation Radicals
n
"
A
1029
M
II I
Flgure 3. (A) ESR spectrum of thymine T cation in 12 M LiCI/D,O, pD 11, lo-' M Fe(CN)Bs, y-irradiated at 77 K, recorded at 145 K, after warming to 170 K. (B) Computer simulation of A, using parameters in Table I, and a line width of 3 G.
A
The Journal of Physical Chemistry, Vol. 85, No. 8, 198 1
Flgure 5. (A) ESR spectrum of orotic acid in 12 M LiCVD'O, pD N 11, lo-* M Fe(CN):-, y-irradiated at 77 K, recorded at 145 K after warming to 170 K. (B) Computer simulation of A, using parameters in Table I, and a varying line width of 5 G in the parallel orientation to 3.5 G in the perpendicualr orientation.
lln
Ii' 1b Figure 4. A ESR spectrum of uracil in 12 M LiCI/D,O, pD = 11, lo-' M Fe(CN))-,) y-irradiated at 77 K, recorded at 145 K after warming to 170 K. (B) Computer simulation of A, using parameters in Table I, and a varying line width of 5 G for the parallel orientation to 3.5 G for the perpendicular orientation.
we employ D 2 0 to reduce line widths, the electron scavengers M Fe(CN)63-and NzO (saturated), as well as basic conditions, pD 11. These conditions reduce the available radiation-produced intermediates to Clz- and smaller amounts of OD.. In Figure 2A the ESR spectrum of y-irradiated 1 2 M LiCl (DzO) with electron scavengers is shown. Figure 2 also shows spectra of y-irradiated 12 M thymine without electron scaM LiCl containing vengers (Figure 2B) and with electron scavengers (Figure 2C). The doublet at g = 2.0036 in Figure 2B arises from the thymine anion.6b This signal is largely removed in Figure 2C by the presence of electron scavengers. Warming samples of y-irradiated 12 M LiCl containing DNA bases resulted in attack of the oxidizing intermediates C12-/OD. on the DNA base. Thus for thymine we observe the spectrum shown in Figure 3A after warming to 170 K. This spectrum arises from the thymine T cation. Unlike the results in the NaC104 glass, there is no evidence for a background signal after warming. This technique then provides the ir cations of DNA bases free from other free-radical species. In Figures 4--7, we show the results of attack of ClZ-/OD. on uracil, orotic acid, isoorotic acid, and cytosine, respectively. These spectra show the anisotropic couplings expected for the ir cations. For example, for isoorotic acid (Figure 6A) only a single anisotropic (10) It has recently been shown that formation of C1,- from .OHoccurs through ClOH- intermediate. At basic pHs the conversion stops at C10H-. Thus, this species may also be present. G. G. Jayson, B. J. Parsons, and A. J. Swallow, J. Chem. SOC.,Faraday Trans. 1,69,1597 (1973); J. Pucheadt, C. Ferrandini, R. Julien, A. Deysine, L. Gilles, and M. Moreau, J . Phys. Chem., 83, 330 (1979).
Flgure 6. (A) ESR spectrum of isoorotic acid in 12 M LiCI/DpO, pD 1 1 , lo-' M Fe(CN):-, y-irradiated at 77 K, recorded at 145 K after warming to 170 K. (B) Computer simulation of A, using parameters in Table I, and a line width of 5.3 G.
Flgure 7. (A) ESR spectrum of cytosine in 12 M LiCI/D,O, pD 11, lo-' M Fe(CN),&, y-irradlated at 77 K, recorded at 145 K after warming to 170 K. (B) Computer simulation of A, using parameters in Table I, and a line width of 5.3 G.
nitrogen coupling is found, whereas for orotic acid (Figure 5A) and uracil (Figure 4A) and anisotropic a-proton coupling and nitrogen are found. For cytosine a more complex spectrum (Figure 7A) arising from two anisotropic nitrogens and an anisotropic a proton is observed. The spectra have been analyzed by use of anisotropic computer simulation programs.'l The best fits to the experimental re(11) Two aniosotropic simulation programs were employed. The first
(R.Lefebve and J. Maruani, J. Chem. Phys., 42, 1480 (1965)) was employed for all simulations except those for uracil, orotic acid, 3-methyluracil, and 6-methyluracil. In these cases it was found necessary to uBe a first-order simulation program (written for this work) which allowed for different line widths as a function of orientation. The line widths were broader for the parallel orientation, as indicated in the figures.
1030
Sevilla et al.
The Journal of Physical Chemistty, Vol. 85,No. 8, 1981
TABLE I: ESR Parameters for Pyrimidine n C a t i o n s radical structure
compd thymine 3
\
N
9
3
OAN/
g value
n
hyperfine splittings, G
gll = 2.0018 A 1 l N = 1 2 gl = 2.0043 A I N = 0 U C H , = 20.2
I1
I1
H
D\Ly~3 I
6-methylthymine
gll = 2.0022 AllN = 9.5 g l = 2.0043 A I N = 0 aCH, = 20.5
0
CH3
I1 811 = 2.0020 AllN = 13.4 g i = 2.0056 A i N = 0
isoorotic acid ;%bhD G
D h k I11
orotic acid
COP-
GAN/
IV
gll = 2.0018 AllN = 1 6 . 0 gl=2.0049 A I ~ = O A x x H= - 8 A y y zf - 24 A,, - - 1 5 gll = 2.0022 AilN = 15.5 gl = 2.0045 A I N = 0
uracil ?~ , D A
Flgure 8. ESR spectrum of 3-methyluracil in 12 M LiCI/D20, pD 11, lo-* M Fe(CN),>, y-irradiated at 77 K, recorded at 145 K after warmlng to 170 K.
N
/
A x x H= - 8 A Y y H= - 24 AZzH= - 15 a
H
V 3-methyluracil
VI 0
6-me thyluracil
VI1
gll = 2.0022 AllN = 10.5 gi = 2.0045 A I N = 0 A x x H= - 9 A y y H= - 25 AztH = - 1 6 gll = 2.0025 AllN = 23.7,
cytosine
9.9
g l = 2.001 g1= 2.0045 VI11
or
AI^ = 0 A --.. x x H= - 5 A Y y H= - 16
AtzH = - 13.5 .3.5
N -%;
A/ IX Same as uracil.
sults are shown in part B of Figures 4-7. The parameters employed in these simulations are reported in Table I. Similar analysis for 6-methylthymine, 6-methyluracil, and 3-methyluracil a cations are also reported. The computer reconstructions were aided by results previously reported for the a cations of thymine,6fi12v13 uracil, 3,5~14and c y t ~ s i n e ~produced ~ J ~ J ~ by photoionization in glasses, in solution, or by y irradiation of single crystals. The parameters from these results were used as starting points and adjusted as appropriate to obtain the best fit. Because of the line width, the results for the a-proton tensor in uracil, orotic acid, and the other pyrimidine cations should be considered approximate. In the simu(12) A. Dulcic and J. N. Herak, J . Chem. Phys., 57, 2537 (1972). (13) W. Flossman, H. Zehner, and A. Muller, 2.Naturforsch. C, 35, 20 (1980). (14) H. Zehner, W. Flossman, E. Westhof, and H. Muller, Mol. Phys., 32,869 (1976). (15) W. Flossman, E. Westhof, and A. Muller, Int. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med., 30, 301 (1976). (16) D. M. Close and W. A. Bernhard, J. Chem. Phys., 66,5244 (1977).
lations for uracil, orotic acid, 3-methyluracil, and 6methyluracil, it was found necessary to employ two line widths as the line width in the parallel orientation was larger (see figures).'l A study of a-cation formation vs. [D+] was performed for thymine in 12 M LiC1. Under basic conditions (9 I pD 5 11)the T cation was formed. At neutral conditions and acid conditions no a-cation formation was found. This suggests that deprotonation at a nitrogen on thymine aids the T-cation formation and stabilization. Assignment of Splittings to Ring Positions. The results found in this work for thymine and our previous work with 5-methylpyrimidines show a large methyl group proton hyperfine splitting of ca. 20 G?cS This indicates unpaired electron density of -0.50 at C-5. For orotic acid, uracil, 3-methyluracil, and 6-methyluracil, the isotropic component of the a-proton tensor (15.7 G) also indicates a large spin density of -0.57 a t position 5. The large nitrogen splitting for all pyrimidine cations except cytosine varies from a low of 9.5 G to 16 G (Table I), reflecting changes in substitution on the ring. The fact that 3-methyluracil (Figure 8) shows no methyl-group coupling verifies the nitrogen coupling arising from the N-1 position, as was previously indicated in our work on N-1-substituted thymine a cations produced by photoionization.6f It is interesting to note that both 6-methylthymine and 6methyluracil ?r cations show significantly lower nitrogen couplings than those found for thymine, and uracil. The results found for the cytosine a cation (Figure 7, Table I) clearly show coupling to two nitrogens. There is also a large reduction of the a-proton coupling. The couplings suggest that the exocyclic nitrogen is a source of one of the couplings. In our previous work, 5-methylcytosine cation was produced at a variety of ~ H S We . ~ ~ reported for the 5-methyl-substituted structure VI11 two smaller splittings of 6 and 11 G for AllN,whereas for 5methyl-substituted structure IX we reported a 28-G A , splitting. The methyl splitting was 20 G for &methy\substituted structure VI11 and 13.8 G for 5-methyl-substituted structure IX. Thus, the large nitrogen AI? splitting and the 11.5 isotropic a-proton splitting found in this work suggest structure IX. However, in our previous work,&the second smaller nitrogen was not observed. In y-irradiated single crystals Flossman, Westhof, and Muller report? splittings similar to those found here except that the second nitrogen splitting was also not observed. McLachlan spin-density calculations clearly indicate that a second AllNsplitting of ca. one-half the larger splitting should be present in structure IX, as is found in those work. Thus, the lack of observation of the second splitting in previous work may be due to a fortuitous overlap of components. Only further experimentation will answer this question. From these results and previous results for the pyrimidine T cations, it is clear that only the cytosine a-cation spin-density distribution is very sensitive to its state of protonation.
The Journal of Physical Chemistry, Vol. 85, No. 8, 7981
ESR Study of DNA Base Cation Radicals
N-1-Substituted Pyrimidines. A number of N-l-substituted pyrimidines were also studied in y-irradiated 12 M LiCl glasses. It was found that N-1-substituted pyrimidines were not very reactive. For example, l-methyluracil, uridine, and cytidine gave no appreciable signal upon warming irradiated glasses. For 1-methylthymine and thymidine, some initial attack was noted, but no increase upon warming was observed. The radical produced in the initial attack was found to be the C-6 OD. adduct (X). This radical results from direct OD. attack and has h
“-32” CA I
D:
k X
been extensively studied before.I7 Its lack of increase on warming may suggest that OD. is scavenged by the chloride ion in 12 M LiCl.’O This indicates that DNA base cation formation in irradiated 12 M LiCl is a result of C1,- not OD. attack. Purine a Cations. The a cations of guanine, guanosine, adenine, and xanthine have been produced upon warming y-irradiated 12 M LiCl D 2 0 glasses. As expected, the purine a-cation ESR spectra are all broad “singlets” with some structure and with g(apparent) 2.0046. For guanine and guanosine, this structure is in accord with that found previously for the guanine and guanosine a cations produced by photoinization.&” The most important result found was that the purines guanine and guanosine formed .~r cations at neutral as well as basic conditions, whereas the pyrimidines tested were not found to do so.
Conclusion The a cations of cytosine,15J6thymine,12J3and uracil14 have been produced previously in irradiated single crystals. These past results are in good agreement with those found here, allowing for differences in matrices. A previous report dealing with hydroxyl attack on pyrimidines in solution assigned what apparently are the a (17) M. D. Sevilla and M. L. Engelhardt, Faraday Discuss. Chern. SOC.,63, 255-63 (1977). (18) J. Holcman and K. Sehested, J. Phys. Chem., 80, 1642 (1976).
1031
cations of thymine (pH ll),uracil (pH l l ) , isoorotic acid (pH 13), and 6-methyluracil (pH 12) to ring-opened radi c a l ~ .This ~ was called into question by Helck6 et al.; l9 however, the alternative radical suggested by these authors was highly unlikely. More recent e ~ p e r i r n e n t a land ~l~~ theoretical2’ work suggests that these radicals are due to a cations, which our work confirms. Our work and the previous work are of importance since combined results we believe unambiquously show that OH. and C12- radical attack on pyrimidines a t basic pHs produces a cations. Our work with purines further shows that C12-and perhaps OH. attack on guanine produce a cations at neutral conditions. This of course has implications for radiation damage to the DNA molecule by the indirect effect. The mechanisms of attack of Clz- and OH. on DNA bases may differ. It is likely that C12- reacts in a simple electron-transfer reaction as in reaction However, in studies of attack of OH. on certain aromatic hydrocarbons and on 4-pyridone, the hydroxyl adduct has been suggested as the precursor to the cation.18,20This work and the work of Bansal and Fe~senden,~ who suggest that in the case of uracil a base-catalyzed dehydration of the hydroxyl adduct results in the T cation, leads to the following mechanism which likely accounts for .Ir-cation formation in thymine. An analogous mechanism would apply to the other pyrimidines studied.
&Y3 AN’kH3 t OH.
0
H
H
-
OH
0
I
H
ti+
H
.-JyH3 Ad 0
H
+
H20
Acknowledgment. We thank the Office of Health and Environmental Research of the Department of Energy for support of this work. (19) G. A. Helck6, R. Fantechi, and M. C. Barbieri, Radiat. Res., 74, 265 (1978). (20) S. Steenken and P. O’Neill, J. Phys. Chem., 83, 2407 (1979). (21) D. C. Chipman, Radiat. Res., in press. (22) K. Hasegawa and P. Neta, J. Phys. Chem., 82, 854 (1978).
