1898
Michael D. Sevilla
(24) The spectra of the vanadyl probe in the frozen solutions of all regions are virtually Identical. (25) Note that the gross dlfferences in the center of gravity of the room temperature spectra when compared with the frozen solution spectrum is due to the difference in cavity frequency from the room temperature and frozen solution experimental arrangements. (26) L. J. Boucher, E. C. Tynan, and T. F. Yen, “Spectral Properties of 0x0vanadium(lV) Complexes. IV Correlation of EPR Spectra with Ligand Type” in “Electron Spin Resonance of Metal Complexes”, T. F. Yed, Ed., Plenum Press, New York, N.Y., 1969, pp 111-130. (27) There is always some reason for concern when low temperature data are used to infer a knowledge of the room temperature situation. In our case, there is the possibility of a temperature induced equilibrium shift from an unbound probe at room temperature to a vanadyl carboxylate (bound probe) complex when the solution is frozen at 77 K. However, comparison of room temperature, isotropic EPR parameters with those computed from the frozen solution, rigid limit parameters are generally In good agreement. See, for example, the tabular data In H. A. Kaska and M. T. Rogers, “Electron Spin Resonance of Flrst Row Transition Metal Complex Ions” in “Radical Ions”, H. A. Kaska and M. T. Robers, Ed., Wiley, New York, N.Y., 1968, Chapter 13, p 598. (28) Ekwall has shown [L. Mandell, K. Fontell, and P. Ekwall, Adv. Chem. Ser., No. 63,89 (1967)] in a similar system, sodium octanoate/decanol/water, that the thickness of the H20 layer ranges from 7 A at the lowest water content to 80 A at the highest water content in the smectic phase D. (29) J. H. Freed, G. V. Bruno, and C. F. Polnaszek, J. fhys. Chem., 75,3385 (197 1). (30) C. F. Polnaszek, G. V. Bruno, and J. H. Freed, J. Chem. fhys., 58, 3185 (1973). (31) S. A. Goldman, G. V. Bruno, and J. H. Freed, J. Phys. Chem., 76, 1858 (1972). (32) S. A. Goldman, G. V. Bruno, C. F. Polnaszek, and J. H. Freed, J. Chem. fhys., 56, 716 (1972).
(33) At low pH, vanadyl exists as the aquated species: VO(H20)5*+. (34) B. Svens and 8. Rosenholm, J. Colloid lnterface Sci., 44, 495 (1973). (35) Admittedly, the Stoke’s law relation treats the tumbllng molecule as a hard sphere, and therefore neglects the effects of iondipole Interactions between the Ionic micelle surface and the solvent, water. These interactions would tend to somewhat increase the value of T~ from this hard-sphere value. However, the effect would be expected to be less than an order of magnitude. (36) The spectra from all other regions were Independent of flat cell orientation. (37) Data on a simllar system, sodlum octanoate/decanol/water L. Mandell, K. Fontell, and P. Ekwall, Adv. Chem. Ser., No. 63, 89 (1967/l, gives the probable reason why region B does not show similar behavlor. This region has a high water content, 72-82%, and therefore the shearing forces are not large enough to produce orientation. (38) Due to the gel-like character of this phase, it was Impossible to prepare a bubble free sample. (39) N. D. Chasteen, R. L. Belford, and I. C. Paul, lnorg. Chem., 8, 406 11969) (40) S:H.-Glarum and J. H. Marshall, J. Chem. fhys., 44, 2884 (1966); 46, 55 (1967). (41) G. Havach, P. Ferrutl, D. Gill, and M. P. Klein, J. Am. Chem. SOC., 91,5726 (1969). (42) P. Ferruti, D. Gill, M. A. Harpold, and M. P. Klein, J. Chem. fhys., 50, 4545 (1969). (43) D. H. Chen and G. R. Luckhurst, Mol. fhys., 16, 91 (1969); Trans. Faraday SOC.. 65. 656 (19691. (44) C. F: Pohaszek and‘J. H. Freed, unpublished results. (45) D. Kivelson, J. Chem. fhys., 35, 156 (1961). (46) G. Meier and A. Saupe, Mol. Cryst., 1,515 (1966). (47) H. Schindler and J. Seelig, J. Chem. fhys., 59, 1841 (1973). (48) The pH is too high for a stable vanadyl carboxylate complex to form in this system.
Electron Spin Resonance Study of Nj-Substituted Thymine T-Cation Radicals‘ Michael D. Sevilla Department of Chemistry, Oakland University, Rochester, Michigan 48063 (Received March 29, 1976) Publication costs assisted by the U.S. Energy Research and Development Administration
a-cation radicals produced by photoionization of thymidine 5‘-monophosphate, thymidine, l-methylthymine, 1-ethylthymine, and thymine in basic 8 M NaC104-DzO glasses at 77 K were investigated by ESR spectroscopy. Analysis of the spectrum of each N1-substituted thymine shows coupling to the 5-methyl group (21 G), to the nitrogen at position 1(All = 13 G, A 1 < 4 G withg. - gll = 0.002), and to (3 protons on substituents at N1. Analyses were confirmed by computer simulations. The “experimental” spin density distribution was calculated and compared to McLachlan MO predictions. Parameters of the interaction of the 1position (3 protons were determined ( p a = 0.21, B2 = 79) and used to evaluate the conformations of the N1 substituent groups. Results found here are compared to previous results in aqueous glasses and single crystals.
Introduction
r-cation radicals2 of DNA bases have been identified in y-irradiated crystalline thymine: t h ~ m i d i n e cytosine: ,~ as well as y-irradiated DNA.6 It is our purpose to aid in the characterization of these radicals and to understand their chemistry by producing them individually in aqueous glasses. In our previous studies the a-cation radicals of several DNA bases were identified and their further reactions ~tudied.~-lO The present work extends the investigation to several N1substituted thymine derivatives (see structure I) and the nucleotide, thymidine 5’-monophosphate (TMP). The Journal of Physical Chemistry, Voi. 80,No. 17, 1976
H R
I. Structure of
thymines in basic
solution
Section 1-Methylthymine and 1-ethylthymine were obtained from
ESR Study of N1-Substituted Thymine a-Cation Radicals
Cyclo Chemical. Thymidine and thymidine 5‘-monophosphate were obtained from Sigma. The same experimental technique as was used in our previous work was employed in this work except that 8 M NaCl04-DzO solutions were used to prepare g l a s ~ e s . ~ In- this ~ technique DNA bases at concentrations ranging from 1x 10-4 to 1X M are photoionized a t 77 K with 254-nm uv light. The photoejected electron is not stable in the NaC104 glass. Those few that are trapped are photobleached with visible light. The C104- acts as an electron scavenger producing a broad “doublet” background signal which hae been attributed to 0-.l1J2 In all cases it was found to be necessary to make the solutions basic (5 X loV2M in NaOH) in order for photoionization to occur. The photoionization process is biphotonic requiring a relatively long lived triplet state.7 It is found that raising the p H increases long lived phosphorescence considerably in these molecules. The increase in phosphorescence is likely associated with the loss of the proton at position 3 (pK = 9 to 10) a t p H values above 10. Computer simulations were performed using programs described previ0us1y.l~The simulations assume axial symmetry for the nitrogen parameters, and are the result of first-order calculations. The proton couplings are assumed isotropic. Gaussian line shapes were used in the reconstructions. Fremy’s salt (peroxylaminedisulfonate) was employed as a standard for g values and hyperfine splittings ( a =~13.0, g = 2.0056).
Results and Discussion a-Cations of Thymidine 5/-Monophosphate ( T M P ) and Thymidine ( T ) .The ESR spectrum of the T M P n-cation radical in a 8 M NaC104-DzO glass at 77 K was moderately well resolved. However, increasing the temperature to 115 K resulted in a significant increase in resolution (Figure 1A). This was found to be the case for the other thymine derivative a cations as well. The phenomenon was found to be reversible and thus is likely due to hindered internal rotation probably of the 5-methyl group. Analysis of the spectrum in Figure 1A yields a large methyl group splitting (21 G), a proton splitting of 8.3 G, and an anisotropic nitrogen whose parameters are reported in Table I. Figure 1B shows a computer reconstruction of the spectrum in Figure 1A based on the parameters in Table I and a line width of 4 G. Accounting for the fact that the broad gll component of the 0- radical overlaps the center of the n-cation spectrum, the fit between experiment and theory is excellent. The broad low field component is the g, component of 0-. The results found for thymidine (Table I) are quite similar to those found for TMP. However, the proton splitting is smaller by 0.5 G and the spectrum was somewhat less resolved at all temperatures. The large splitting in the T M P cation due to three equivalent protons is clearly due to the methyl group at position 5. The nitrogen splitting could arise from position 1 or 3. Spin density calculations for these cation radicals performed by a number of methodssJ4 clearly show position 1to be the site of greater unpaired spin. Results presented below for 1methylthymine verify this. This leaves only the splitting due to a single proton unassigned. This splitting could arise from 6 position or the 6 proton on the ribose group a t position 1. 1-Methylthymine ( I M T ) and 1-Ethylthymine (1ET). To test the possible interaction of the ribose group hydrogens with the pyrimidine ring two model compounds (1MT and 1ET) were investigated. At 77 K in basic 8 M NaC104 the
1899
Figure 1. (A) The first derivative ESR spectrum of the thymidine 5’monophosphate a-cation radical in a basic 8 M NaCIO4-D20 glass at 115 K, produced by photoionization. The photoejected electron reacts with C104- to produce 0-.The 0-spectrum is a “doublet” which accounts for the broad low field component and the central broad underlying component. (B) Computer simulation of the a-cation spectrum in A employing parameters in Table I and a 4-G line width.
spectrum of the 1MT a cation was poorly resolved. Best resolution was found a t temperature of 140 K and above. The spectrum a t 145 K (Figure 2A) clearly shows coupling to two methyl groups. Figure 2B shows the last three components at three times the gain. Analysis of these spectra yields the parameters in Table I. The reconstruction shown in Figure 2C is based on these parameters and a line width of 3 G. The line positions of the major components are well accounted for. Two smaller components which are resolved in the simulation are not observed in the experimental spectrum. Considering the approximation of constant line width used in generating the reconstruction the overall fit is considered sufficient to verify the analysis. The assignment of the two methyl group splittings is obvious from the structure of 1-methylthymine. The fact that a 8-G splitting is observed for the methyl group at position 1 and that no further proton splitting is observed from the 6 position supports the suggestion that the 8-G splitting found in T M P is due to the ,8 proton on the ribose group. The spectrum found for 1ET at 145 K was not as well resolved as for 1MT. However the spectrum was sufficiently resolved to observe the major splittings (see Table I). Only one proton coupling of the ethyl group was sufficiently large to be observed (6.5 G). The other may have contributed to the increased line width observed for this species. The results for the a cation of thymine in 8 M NaC104 are included for comparison to our results in other glasses.7~8In our previous work it was found that the a-cation splittings were not sensitive to changes in protonation or matrix. We find this to be the case in NaC104 as well. The results in the table are within experimental uncertainties of those found previously in 8 M NaOD, 5 M K2C03,and 6 M D3P04glasses. It should be noted that the resolution found for the thymine a cation places an upper limit to the magnitude of the 6H splitting of approximately 3 G. This result is in accord with those found for TMP, T, l M T , and 1ET. In addition recent experiments with 5,6-dimethyluracil in NaC104-DzO glasses show no 6-methyl splitting for the a cation. The experimental line width of 3 G limits the magnitude of the splitting to be less than 2.5 G.
Spin Density Distribution Calculations of the spin density distribution in the N-substituted thymine a cations by the McLachlan methods show that the spin density is localized mainly at positions 1( p l l =
. The Journal of Physical Chemistry, Vol. 80, No. 17, 1976
Michael B. Sevilla
1900
TABLE I: T-Cation Radicals of Thymine Derivatives in 8 M NaC104-Dz0 Glasses Compound
Temp, K
Thymidine monophosphate Thymidine 1-Methylthymine 1-Ethylthymine Thymine Thymine single crystala (anhydrous)
115 145 145 145 145
a
77
G 21.3 21.4 21.1 21
20.3 19.4 f0.5
_-___--
&cH,G
A1lN,G
8.3 7.8 8.4 6.5
13.1 13 13.0 13
~k0.3
13.2 f0.5
11.9
AIN,G
