J . Phys. Chem. 1993, 97, 3401-3408
3401
The Protonation State of One-Electron-Reduced Cytosine and Adenine. 1. Initial Protonation Sites at Low Temperatures in Glassy Solids J. Barnes and William A. Bernbard’ Department of Biophysics, University of Rochester, Rochester, New York 14642 Received: October 14, 1992; In Final Form: December 15, 1992
Electron paramagnetic resonance spectra of cytosine and adenine, one-electron reduced in a 9 M LiCl glass at 4 K, show that in each case protonation occurs at the amino group. In order to understand this, we produced a number of one-electron-reduced cytosine and adenine analogues in different glassy environments, at 4 K, and used Q-band EPR to determine the sites of protonation. We conclude that the specific association of Li+ with the ring nitrogen blocks protonation at the expected sites, N 3 of cytosine and N1 of adenine, diverting protonation to the less basic amino group.
Introduction The radiation chemistry of DNA is initiated by excitation, superexcitation, one-electron oxidation, and one-electron reduction. The majority of the irreparable damage is expected to come from clustered damage,’ what Ward has termed ‘locally multiply damaged sites”.2 There are two possible roles one-electronreduced bases may have in this model: one-electron reduction alone is known to inactivate single-stranded phage DNA with a low efficiency;’ alternately, the trapping of an electron on a base may decrease recombination, leading to higher initial free radical and radical product yield^.^ The EPR spectra of irradiated DNA and oligomers from 4 to 77 K clearly indicate the presence of one-electron-reduced bases. Further, there is evidence that the distribution of reduced bases is not uniform, but favors the pyrimidines over the purines, and depends upon the local en~ironment.~-* There are four possible mechanisms to explain this skewed distribution: (1) selective formation, in which dry electrons, originating from DNA and its surroundings, are preferentially captured by specific bases; (2) paramagnetic mobility, in which the electrons migrate from an initially random distribution to specific trapping sites; (3) differential recombination, where certain radicals are more stable toward initial, intracluster recombination than others; and (4)at high dose, saturation asymmetry, where certain radicals are more stable than others against recombination due to intersectingtracks. The first three mechanisms influencethe Gvalues of each reduced base, and the fourth mechanism influences the destruction constant^.^ (The G value is defined as the number of radicals produced per 100-eV absorbed dose, and the destruction constant is defined as a fraction of radical concentration lost per unit absorbed dose.) It must be emphasized that all fourpossibilities can lead to the same result at high dose, namely, an asymmetric distribution of one-electron reduction for any given base sequence. Paramagnetic mobility is most often assumed to explain the distribution of electron adducts found in irradiated DNA and oligomer systems. Experimental work has identified three important factors for this model: 1. Electron Mobility in Stacked DNA Bases. There is now ample evidence that electron transfer between a reduced base and another base in a stacked system can occur.IG12 From measurements of microwave absorption in irradiated dry DNA, an upper limit on the mobility, p < lo-* m2 V-I s-I, or on the product of the mobility and lifetime, p7 < 5 X lO-I5 m2 V-I, of the induced charge carriers was found.I3 While the range of electron transfer has been suggested to be from 20 to as great as
* To whom correspondence should bc addressed.
200 stacked bases,I1.I2a range of > A > G.I5 However, recent measurements of the RP of the pyrimidine bases found that of C to be similar to that of T and U, --l.l V/NHE, at 8 < pH < 9.16 The higher than expected value for C is explained by the extra driving force provided by the rapid protonation of the reduced anion. This brings us to the third important factor. 3. Protonation of a Reduced Base, Returning It to Its Initial Chargestate. The result of pK, measurements of theone-electronreduced bases in solution suggests that, upon reduction in a doublestranded DNA environment, cytosine and adenine will protonate from their base pairing partners, while thymine and likely guanine remain radical anions.” There is also evidence that the presence of extended, hydrogen-bonding networks in the solid state can selectively increase the total yields of certain This effect is postulated to be due to the ability of the network to rapidly separate the charge change, +1 for oxidation and -1 for reduction, by >20 A from the radical. Thus, the ability of a reduced base to protonate depends both on its pKa and the protondonating environment. At present, these three concepts are able to predict with some success the distribution of reduced bases in a given DNA model environment. However, the existence of these factors do not necessarily imply that paramagnetic mobility is the only mechanism by which the electron distribution is skewed. Currently, there is no compelling evidence, to our knowledge, to completely exclude the other three mechanisms. One approach to better understanding these processes is the deconvolution of EPR spectra of reduced oligomer sequences in various environments at low temperatures. Toward this goal, it is important to understand the EPR spectra of the reduced DNA bases in high-salt glasses such as 9 M LiCI.*O In this paper, we present data that helps explain the polymorphic nature of reduced cytosine, and we observe that reduced adenine displays analogous properties. We will argue that the polymorphism in both adenine and cytosine is the result of Li+ association with the ring nitrogen of both bases. We then discuss the implicationsfor deconvolution of the EPR spectra of reduced oligomers and irradiated DNA, in various environments, including LiCl glasses.
0022-3654/93/2097-3401$04.00/00 1993 American Chemical Society
Barnes and Bernhard
3402 The Journal of Physical Chemistry, Vol. 97, No. 13. 1993 Experimental Section
All cytosine and adenine bases, nucleosides, nucleotides, and base analogues, were purchased from Sigma Chemical Company, and used without further purification. For theglasses, CsF, optical grade, was purchased from Sigma Chemical Company; LiCI, anhydrous ultrapure, was purchased from Alfa Products; NaH2PO,.HOH, analytical reagent grade, MeOH, optical grade, and DOD, 99.96% minimum isotropic purity, were purchased from Aldrich Chemical Company; NaD2P04,98.8% isotropic purity, was purchased from MSD Isotopes; MgCly6H20, analytical grade, was purchased from Mallinckrodt; and A1Cl3.6H20,purum grade, was purchased from Fluka. All were used without further purification. Stock solutions were prepared with filtered, deionized, and distilled water from a Penpure Niagara Incorporated system. Stock solutions of 7 M NaH2P04,13 M LiCl, 9.6 M CsF, 4.5 M MgCI2,and 5 M AlCl,, were prepared and used throughout. The bases were first dissolved in HOH, under air, then an appropriate amount of the high-salt solution was added, to reach the final quoted concentrations for each glass. All solution showed no sign of precipitation a t room temperature, and appeared to the eye to be clear glasses upon cooling to 4 K. All the cytosine and cytosine analogue concentrations were 50 mM except for the MeOH glass, which was 200 mM, and theoligomers, which were < l o mM. The adenine and adenine analogue monomer concentrations varied between 20 and 50 mM, depending on their solubility except the 1:1 Me0H:HOH glass, which used 200 mM 5'-AMP. Theadenineoligomerswere a t 3.0 pH units.
+
V
3.0 mT
Figlw3. Q - h n d EPR spectraof 3'-UMP, top, andNfl-dimethylcytosine, bottom, one-electron reduced in a 9 M LiCl glass at 4 K.
doublet spectra upon reduction. The absence of a significant &hyperfine coupling, or any notable broadening, of the doublet spectra in Figure 3 implies that protonation at the C4 group probably does not occur. Under theassumption that the chemistry of the base is not otherwise significantly altered except at the C4 group, these spectra eliminate other possible protonation sites. The most energetically favorable site for protonation of the reduced cytosine ring is expected to be N3. It is known that, upon one-electron reduction, the (BrBnsted) basicity of N3 of cytosine increases >9 pH units.I7 However, experimental results24.25 and theoretical calculations26have shown that this site also binds Li+, which should block protonation at N3 in the glassy state, after one-electron reduction. The amino group is also expected to increase in basicity upon reduction. As a result, the C(N3-*Li+)*-radical protonates at the amino group to form the C(N3-.Li+,N4+H)* radicalaZ0 KNOH I
R C(N3-Li +)
-
C(N3.-U+,NI+H'~
This effect is also seen in an acidic 9 M LiCl glass ([H+] = 10-0.2) and in a 5 M NaH2P04glass ([H+] = 10-3.0),in which 5'-dCMP is initially protonated at N3. Upon reduction at 4 K,
+
+
R C(NJ+H)
C(NS+H,NQ+H) ORTHOGONAL ROTAbfER +
In glasses containing no ions that associate strongly with N3
of cytosine,and in which the cytosineis not protonated, we predict that the EPR spectrum of reduced cytosine should be a doublet. This is because the larger Cs+ ion, with an ionic radius of 1.69 A, is expected to associate less strongly with cytosine than the smaller Li+.25327In Figure 4 parts C and D,reduced S'ICMP is shown in a 9 M CsF glass ([H+] = 10-11.4),at X-and Q-band, respectively. Although the CsF glass acts as an inhomogeneous broadening agent, it can be seen from the X-band spectrum that one-electron-reduced Y-dCMP gives a doublet, which suggests that either the C'- or C(N3 + H)' radical is produced. In contrast, the smaller cations with higher charges, Mg2+and A13+,should associate strongly with N3 of cytosine and divert protonation to the amino group upon one-electron reduction. Figure 4 parts E and F show reduced 5'-dCMP in a 4.5 M MgClz glass ([H+] = l W 7 ) and a 5 M AlC13 glass ([H+] = 100.0), respectively. The triplet nature of these spectra indicate that the C(N3-.Xq+,N4+H)* radical was produced. An environment expected to allow protonation at N3 is the MeOH/HOH glasses. Figure 5A shows the X-irradiated, photobleached 9:2 (by volume/volume) MeOH/HOH glass, containing 10 mM K3Fe(CN)6as an electron trap. The EPR spectra of the Fe3+ and Fez+ ions are very broad at Q-band, so only the *CH20Hradical is readily apparent. Figure 5B shows the result of using 200 mM 5'-dCMP as the electron trap. The outer portion of the 'CH20H spectrum in Figure 5A was shifted and scaled so as to match that portion of the spectrum marked
-
Barnes and Bernhard
3404 The Journal of Physical Chemistry, Vol. 97, No. 13, 1993
4%3.0 mT
F i p r e 5. All EPR spectra were recorded at Q-band at 4 K. (A) 9:2 volume/volume McOH/HOH glass, with IO mM K3Fe(CN)6 as an electron trap, X-irradiated at 4 K and then photobleached for 10 min witha MikrarkIlluminator 100-Warclamptoremovethetrappedclectron signal. (e) 9:2v/v MeOH/HOH glass with 200 mM 2’-dC as theelectron trap, X-irradiated at 4 K and then photobleached as above. (C) The result ofsubtracting thefirst spectrum, shiftedandscaledsoas tominimize the sum of the squares of the differences, over the marked portion of the second spectrum. The shift was 1 data point to the left, and the scale was 1.003.
Figure 6. Q-band EPR spectra of (A) C. (B) 2’-dC, (C) 5‘-dCMP, and (D) 3’-CMP, all in a 9 M LiCl glass, one-electron reduced at 4 K, annealed to between 100 and 120 K for IO min, then returned to 4 K.
.
-5 0
TABLE I: TOWWidths, Measured in Peak-to-Perk of the First Derivative EPR Spectra, of Cytosine Derivatives, One-Electron Reduced at 4 K in Various Clrsses. ”be Widths Have an Accuncy of fO.1 mT solute cytosine 2’-deoxycytidine 5’-dCMP 3’-CMP p(dC)r p(dC) I O 3’-UMP N4,N4-dimethylcytosine S’ICMP cytosine I -methylcytosine 5‘-dCMP 2’-deoxycytidine
glass width (mT) 9 M LiCl 2.9 9 M LiCl 2.8 9 M LiCl 3.0 9 M LiCl 2.9 9 M LiCl 2.3 9 M LiCl 2.5 9 M LiCl 2.1 9 M LiCl 2.2 9 M LiCl 0.7 M HCI 3.0 5 M NaH?POr 2.9 5 M NaH?PO, 3. I 5 M AlCli 4.4 9 MeOH/2 HOH 2.6
+
type triplet triplet triplet triplet doublet doublet doublet doublet triplet triplet triplet triplet doublet
in Figure 5B,using a least-X2criteria. The difference spectrum, clearly a doublet, is shown in Figure 5C. The fact that the doublet is well resolved means that no less than 2/3 of the radicals are in the C’- or C(N3 H)’ state. We now return to the protonation state of the reduced cytosine oligomers, which are 2.4-mT doublets, where the total spectral width is measured from the low-field “up” peak to the high-field “down” peak of the first derivative spectra. These widths are used only as a relative measure of the total coupling present in a radical and are considered to be accurate to within fO.l mT. The widths of a series of cytosine analogues, reduced at 4 K in a variety of environments, are collected in Table I. The cytosine analoguesreduced in a neutral LiCl glass at 4 K,which correspond to the C(N3--Li+,N4+H)* radicals, have widths of 2.8-3.0 mT. The width of N4,N4-dimethylcytosine in LiCl, 2.2 mT, probably represents the width of the radical anion, since it is blocked from protonation at N3 by Li+ and is not protonated at the amino site, as evidenced by the lack of a resolved &hyperfine coupling. The extra amino proton couplings of 0.8 mT, 0.5 mT of which is the major @ coupling, can be used to estimate the spin density at C4, by the relation aPIS,= p(C4)82 cos2 8. Using the *CH*NH,+ radical as a model,28and assuming a rapid rotation of the amino group, we find €4 1.9 mT, which gives p(C4) = 0.26, similar to the value of p(C4) = 0.32 reported by Symons et al. for the same radical at 77 K.23 From the acid glasses which yielded triplet spectra, we find that the width of C(N3+H,N4+H)*+ is
+
-
i
f
@
0
20
40
69
80
A n n e a l i n g temDeratlre,
100
120
’40
K
Figure 7. increase in the total spectral width of the Q-band EPR spectra, as recorded at 4 K, as a function of the annealing temperature. The one-electron reduced solutes are C (A),2’-dC (a), 5’ICMP (0),and 3’-CMP (V),in a 9 M LiCl glass, and Y-dCMP, one-electron reduced in a 4.5 M MgC12 glass (A)and in a 5 M AICI, glass (H). All samples were annealed for 10 min before returning to 4 K to record the spectra. The error in the annealing temperature is i 2 K. The error in the spectral widths is h0.2 mT.
2.8-3.0 mT. However, in the AlCl, glass, the width is larger, at 4.4 mT. (We shall return to this result in the Discussion.) Comparison of the C(N3+H,N4+H)*+ radical’s width, from the acidic LiCl glass, with that of the C(N4+H)* radical shows that the extra coupling from the N3 proton must be small. The isotropiccouplingto H(N3) for the C(N3+H)’ radical in a crystal of cytosine monohydrate was found from ENDOR to be 0.2 mT.Z9 Finally, the reduced cytosinewidth in the MeOH and CsFglasses, 2.6 mT, is assumed to be the width of C(N3+H)*. It is seen, then, that the width of the reduced cytosine oligomers, 2.4 mT, suggests that they remain predominantly unprotonated at 4 K in a 9 M LiCl glass. Shown in Figure 6 are the EPR spectra of the same systems in Figure 2, after annealing to 120K for 10min and then recooling to 4 K. The spectral widths, plotted as a function of annealing temperature in Figure 7, have increased to -3.5 mT, with a concurrent increase in the resolution of the triplets. Also shown are the spectral widths of one-electron reduced 5’-dCMP in the MgCI2 and AICl, glasses, upon annealing. This increase is significant and needs to be explained. We do not believe that the geometry of the protonated amino group is relaxing from a constrained position into an sp3 configuration. This explanation would have the amino group approach a tetrahedral geometry, increasing hyperconjugation to the @ proton, but also creating two new @ couplings of the size of the major @ coupling. Powder simulations indicate that this will decrease, not increase,
One-Electron-Reduced Cytosine and Adenine
The Journal of Physical Chemistry, Vol. 97, No. 13, 1993 3405 TABLE 11: Total Spectral Widths, Measured in Peak-to-Peak of the First Derivative EPR Spectra, of Adenine Derivatives, One-Electron Reduced at 4 K in Various Classes. The Widths Have an Accuracy of hO.1 mT molecule
glass
width (mT)
type
J Figure 8. Q-band EPR spectra of adenine analogues,one-electron reduced at 4 K in a 9 M LiCl glass. From top to bottom: ( A ) adenosine, (B) 5'-AMP, (C) 3'-AMP, (D) 3',5'-cyclicAMP, (E) p(dA)d,and (F)p(dA),o.
9-methyladenine adenosine 5'-AMP 3'-AMP 3'-AMP 3',5'-cyclic AMP p(dA)d p(dA)u p(dA)iu 5'-IMP N6,N6-dimethylade,iine N6,N6-dimethyladenosine 6-chloropurine riboside adenosine 5'-AMP p(dA)r 5'-AMP 5'-AMP 5'-AMP
9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCI/DOD 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl 9 M LiCl + 1.3 M HCI 5 M NaH?PO, 5 M NaH2POd 9 M CsF 9:2 MeOH/HOH 1:l MeOH/HOH
2.0 2.2 2.1 2.2 0.9 2.2 2.3 2.1 2.3 1.4 1.5 1.4 1.4 1.7 1.7 1.8 1.8 2.2 2.0
singlet singlet doublet doublet singlet doublet doublet doublet singlet singlet singlet singlet singlet singlet singlet singlet singlet singlet singlet
the triplet resolution. This leaves us with two hypotheses. One is that the protonation state of the radicals shifts upon annealing. The second explanation is that the cation association with the cytosine ring can increase upon annealing the glass, resulting in a stabilization of spin density at the C 4 C 6 carbons. Both hypotheses are examined below in more detail. There are several ways the protonation state of the reduced cytosine rings could shift with temperature. If a mixture of C(N3--Li+)'- and C(N3-.Li+,N4+H)* radicals exists at 4 K, the resolution of the triplet spectrum is decreased. Upon annealing, the C(N3.-Li+)*- radicals protonate at N4 and the triplet width and resolution increase. It is less probable that the radicals are converting from C(N 3-.Li+ ,N4+H)* to C(N 3+H,N4+H)*+, since the width of the second radical, from the acidic LiCl glass, was no broader than that of the first, at 4 K. (In some of the acidic glasses, annealing leads to a conversion of the C(N3+H,N4+H)*+radical into an altogether different product, which will be the subject of a later report.) Conversion of C(N3.-Li+,N4+H)* to the C(N3--Li+,N4+H,02+H)*+ radical is also a possibility. Protonation of reduced cytosine at either N3 or 0 2 increases the spin density at C4, while decreasing it at C6, which would increase the triplet resolution. (See ref 30 for INDO calculationson thevarious protonated, one-electron-reducedforms of cytosine, providing some indication of expected shifts in spin density.) Whatever protonation reaction is assumed, it must have a large range of activation energies, since the spectral width increases only gradually from 40 to 120 K (Figure 7). The other hypothesis for the increase in spectral width upon annealing is more compelling. The positive ion associated with either N3 or 0 2 , because of the increased polarizability and negative charge density of the reduced cytosine ring, shifts closer to the ring upon annealing. The postulated effect of this stronger association is that a larger negative charge density is stabilized on the N3-02 side of the cytosine ring and the unpaired electron density increases on the C 4 4 6 side of the ring, leading to increased coupling to H(C6) and H(N4) and, thereby, anincrease in both triplet resolution and spectral width. The AI'+ ion is already stronglyassociated with thecytosine ring before reduction, while the Mg*+and Li+ ions apparently shift some upon annealing (Figure 7). We will come back to this point in the Discussion. Adenhe. Shown in Figure 8 are the EPR spectra of adenine, adenosine, 5'-AMP, 3'-AMP, 3',5'-cyclicAMP, P ( ~ A ) and ~, p(dA)lo, all reduced in an 9 M LiCl glass at 4 K. They all have nearly the same total width, 2.1-2.3 mT, as can be seen in Table 11. However, the phosphate monomers appear to resolve a coupling that is not as apparent in adenine, 9-methyladenine(both are singlets like adenosine), adenosine, or the oligomers. It can also be seen that the EPR spectrum of reduced adenine in an
oligomer is length dependent, resolving the extra coupling better for shorter chain lengths. The resolution of this extra coupling is probably dependent upon the degree of stacking that occurs in solution: adenine, 9-methyladenine, and the longer length oligomers are better stackers than the nucleosides and nucleotide~.~'~~~ Again, thequestion arises as to the sourceof the extra coupling, and by analogy with cytosine, whether or not it is caused by a specific interaction with the ions in the LiCl glass. Although Li+ does not associate with adenine in a 1 M LiCl solution at room temperature, CI- does associate with the amino group, possibly as a hydrogen-bond acceptor.24 The result would be to lengthen the N-H bonds, thus increasing the partial negative charge on the amino nitrogen. This interaction may lead to the amino group becoming the better proton accepting site, upon reduction of the adenine ring. It should be noted that the ion associationconstants from ref 24 were measured using the increase in T Iquadrupolar relaxation times of the 'Li and nuclei, when bound in an asymmetric ion-base complex. If the exchange rate of cytosineassociated Li+ with Li+(aq) is >lOS/s, then Li+ association with cytosine would be missed. Even if the degree of association is small at standard concentrationsand temperatures, it will increase when going to very high ionic strengths and low temperatures. Thus, Li+ might play a role for adenine similar to that for cytosine. In order to experimentally determine the site of the extra coupling, the amino group was replaced by a keto oxygen, a chlorine, or a dimethylamino group. Figure 9 shows the resulting EPR spectra upon reduction at 4 K. In all cases, 1.4-mT singlets are observed, which we assume represent the unprotonated, reduced adenine radical. As with cytosine, the lack of an observed @protonsplittingindicates that theaminogroupisnot protonated. With the assumption that the various substituents do not greatly alter the electron distribution of the adenine ring, we conclude that the extra coupling in one-electron-reduced adenine is due to protonation at the amino group. The behavior of some adenine analogues, reduced in a variety of environments, is instructive. The EPR spectrum of reduced adenosine in an acidic LiCl glass ([H+] = 10+0,') is shown in Figure 10A. The EPR spectra of reduced 5'-AMP and reduced P ( ~ A in ) ~5 M NaH2P04glass ([H+] = 10-3.0)are shown in Figure 10 parts B and C, respectively. Like the cytosine derivatives, in these acidic glasses A is protonated at N1 before reduction. Unlike reduced C, reduced A does not further protonate at the amino group, which is consistent with the smaller change in pK, of 8.6 pH units upon reduction of An1' The corresponding change for cytosine is >9 pH units." The spectral width of the N1-protonated, reduced adenine ring is found to be 1.7 mT.
D
;o
)'
J/
*----/'
Ill1
3406 The Journal of Physical Chemistry, Vol. 97, No. 13, 1993
A"
B
------I
L
D
-
3 0 mT
Barnes and Bernhard
-
/I\
r\
'r
Figure 9. Q-band EPR spectra of modified adenine analogues, oneelectron reduced at 4 K in a 9 M LiCl glass. From top to bottom: (A) 5'-IMP, (B) 6-chloropurine riboside, (C) N,N-dimethyladenine, and (D) N,N-dimethy ladenosine.
1
3.0 mT
Figure 11. The result of subtraction of the MeOH background spectrum, shown in Figure 5A, from (A) 9:2 MeOH/HOH glass with 100 mM 5'-AMP as an electron trap, and (B) 1:l MeOH/HOH glass with 200 mM 5'-AMP as the electron trap. All glasses were pbotobleached 10 min with a Mikrark Illuminator 100-W arc lamp after X-irradiation at 4 K. The MeOH background spectrum was fit to the low-field side 'CHIOH features, using the same range as that shown for Figure 5B.The x 2 minimized fitting parameters were found to be as follows: (A) scale = 1.002, shift = -1, and (B)scale = 0.58,shift = 12.
adenine protonates at the N1 site to give the A(Nl+H)* radical, which has been observed in the solid ~ t a t e . 3 ~ H
-3-
k
-
3.0 mT
Figure 10. Q-band EPR spectra of adenine analogues, one-electron reduced at 4 K in various environments. From top to bottom: (A) adenosine, in a 9 M LiCl 1.3 M HC1 glass; (B) 5'-AMP in a 5 M NaHzP04 glass, (C) p(dA)4 in a 5 M NaH2P04 glass, and (D) 5'-AMP in a 9 M CsF glass.
+
A(Nl+H)
R A(N ~..U+,NB+H)
-
Upon annealing the reduced adenine in the LiCl glass, the A(Nl-.Li+,N6+H)* converts to a carbon-protonated radical. Such protonations have been demonstrated in the solid statd6 and in aqueous solution.37 The identification of this radical will be the subject of a later report. Discussion
Reduction of 5'-AMP in the 9 M CsF glass yields a 1.8-mT singlet (Figure 10D). Thespectralwidthsuggests that this radical is probably the N1-protonated, reduced adenine. As before, we assume that the ions of this glass form weaker associations with the adenine base than those of LiCl. We then conclude that the specific associations of the Li+ and C1- ions with adenine are responsible for the shift in the protonation site upon reduction. In Figure 11, the result of the subtraction of the MeOH oxidation spectrum from reduced 5'-AMP in both 9:2 and 1:l MeOH/HOH glasses is given. Both spectra are 2.1-mT singlets but show very slight inflections. Theinflections may be the result of stacking of 5'-AMP at the higher concentrations used,31J* or the formation of cyclic, hydrogen-bonding adenine pairs in the more hydrophobicenvironmentof the methanol glass.34 However, it is more likely that the observed inflections are the result of incompletesubtraction of the MeOH background.35 Regardless, the MeOH glass does not yield a doublet comparable to that observed in the LiCl glass (Figure 8C) and therefore is likely to represent the N I-protonated, reduced 5'-AMP. From the data above, we conclude that the Li+ ion associates, at high concentrations, with the preferred protonation site of adenine, N1, and probably N3 and N7 as well. Upon one-electron reduction, adenine then protonates at the amino group, to give the A(Nl-.Li+,N6+H)' radical. When the site is not blocked,
The Li+ blocking hypothesis is consistent with the recently published data of Hiittermann.Z2 It explains, for example, why the X-band spectrum of cytosine, reduced at 77 K in a BeF2 glass, is a triplet, but reduced cytosinein a glucose glass yields a doublet (from Figures 5 and 4, respectively, of ref 22). The BeZ+ ion, with an ionic radius of 0.31 A, has the potential to associate to a greater degree with N3 of cytosine than Li+ does, which will shift the protonation site to the amino group. The glucose glass, like the methanol glass, is a hydrogen bond acceptingand donating glass a n d is not expected to block protonation sites on the cytosine ring. Hence, reduced cytosine protonates at N3, and no extra proton coupling is resolved. Our data also agree with that of Symons et al., except for the reduction of C analogues in a 9:2 MeOH/HOH glass.23 Triplet X-band spectra were found at 77 K,after X-irradiation and annealing to 130 K to selectively remove the methanol oxidation products. It was this result which suggested that the Li+ blocking hypothesis was wrong. We have found reduced 2'-dC to be a doublet at 4 K in this same glass. Upon annealing this system to 133 K,a series of complex spectral changes occur, ending in a triplet which occurs only in the presence of a cytosine analogue. However, this radical power saturated at -20 mW at 77 K, which is higher than the -0.1 mW expectedfor theone-electronreduced base. It seems likely that other radical reactions are
One-Electron-Reduced Cytosine and Adenine occurring upon annealing, but we did not pursue further the identification of this radical. The physical mechanism by which the C(N4+H)' radical is produced is still uncertain. Symons has proposed that the high ionic strength stabilizes the amino-protonated form over the C(N3+H)* form. Presumably, this is because, upon freezing, the salt anions predominate at the amino and C5=C6 double bond, while the salt cations predominate at the 0 2 and N 3 atoms, due tosimpleelectrostaticconsiderations: the large dipole moment of cytosine, -8.0 D,3s induces an equal and opposite dipole in its environment. Upon reduction, the zwitterionic-like C(N4+H)' radical might then be preferentially stabilized. This would require the solvation enthalpy of cytosine in 9 M LiCl to be on the order of the difference in energies of the C(N4+H)* and C(N3+H)' radicals, 2 eV,16which seems high, even for a 9 M LiCl solution. It also does not explain why the C(N3+H)* radical was formed in the 9 M CsF glass. Finally, we expect that, as the charge on the positive ion increases in the series from Li+ to Mg2+to A13+, the ratio [C(N4+H)*]/[C(N3+H)'] should also increase. However, it is seen from Figure 7 that the increase in spectral width did not follow a simple pattern. For these reasons, we prefer an explanation involving specific associations of Li+ and Cl- with the cytosine ring: C1- accepts hydrogen bonds from the amino group, increasing the basicity of this group, while Li+ accepts a "lone pair" from N3, sterically preventing protonation at N3. The shorter adenine oligomers show considerable doublet character, indicating that reduced adenine is protonating at the aminogroup. As the chain lengthens, the spectral width remains constant, but the resolution decreases. Although the spectrum of reduced p(dA)lo is a singlet, the width argues that a certain fraction of the adenines are protonated at the amino group. The reduction in resolution argues that a growing fraction of reduced adenines are not protonated at the amino group, but it is not known whether they are N1-protonated or remain true radical anions. In the LiCl glasses, why does reduced adenine in p(dA)lo protonate at the amino group, while reduced cytosine in p(dC),o does not? We suggest that the explanation comes from the conformation of the two oligomers. For example, poly(C) and poly (A) adopt single-stranded,right-handed helical conformations in solution. Poly(C) is the tighter structure, with a pitch of 18.7 A and 6 nucleotides per turn. Poly(A) has a pitch of 25.4 A, with 9 nucleotides per turn.32 An examination of these structures shows that the amino group of adenine in poly(A) is more accessible tosolvation, and thus protonation, than that of cytosine in poly(C). The ring nitrogens, N 1 of adenine and N 3 of cytosine, both appear to be accessible to solvation and, presumably, Li+ association. These results help interpret some anomalousresults from earlier deconvolutions of one-electron-reduced oligomers in 9 M LiCl glasses at 4 K.9 A significant difference in the calculated electron distributions on the oligomers was noted for the single stranded A-T oligomers. The electron distribution appeared to shift in favor of adenine, in going from the HOH to the DOD glasses. This is probably an artifact due to the benchmark spectrum chosen for reduced adenine, a 2.1-mT singlet. The width suggests that a fraction of this spectrum is due to the amino-protonated form of reduced adenine. The DOD results, which are not as sensitive to the protonation state of reduced adenine, are more likely to reflect the actual distribution of electron attachment in the oligomers. The amino protonation site would seem to be of little importance for the case of an isolated, one-electron-reduced base in a duplex DNA environment. Although the B-DNA helix, with an average pitch of 33.8 A and 10 nucleotides per turn,3* more closely resembles the open poly(A) structure, protonation at the amino group is not expected because of the presence of stronger acids hydrogen bonded directly to the bases: guanine in the case of cytosine, and thymine in the case of adenine.I7 However, in a spur, a proton shift across the double strand does not remove the
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The Journal of Physical Chemistry, Vol. 97, No. 13, 1993 3407 excess negative charge from the neighborhood of the reduced base, lowering the G value for the base radicals through a differential recombination mechanism. The excess -1 charge could be removed by protonation of reduced C or A at the amino group, from water in the major grove. A fraction ofthose reduced adenine and cytosine bases that survive initial recombination in irradiated DNA may thereby be amino protonated. Conclusions
In neutral, protic glasses such as the methanol/water glasses, one-electron-reduced cytosine is protonated at N3, while oneelectron-reduced adenine is protonated at a ring nitrogen, presumably N 1. Specificion associations can shift the protonation sites, and the spin densities, of the radicals. Cation association with N3 of cytosine or N1 of adenine, as in the neutral, 9 M LiCl glass, results in the amino-protonated, reduced forms of both bases. Protonation of cytosine prior to reduction in the acidic 5 M NaH2P04glass ([H+] = O) resulted in the N 3 and aminoprotonated radical, but for adenine, a second protonation did not occur. Thus, at [H+] > 10-3.0M, the electron adduct of cytosine is doubly protonated (i.e., is a radical cation), while adenine remains neutral for [H+] < lo4 M. The spin density at C4 and C6 of one-electron-reduced cytosine is larger in the AlC13 glass, than that in the MgCl2 and LiCl glasses, after reduction at 4 K, apparently due to a stronger association of the A13+ion with the cytosine ring. Upon annealing, the Li+ and Mg2+ions move in closer to the N3 or 0 2 atoms and consequently stabilize the lone pair electrons on N3 and 0 2 , while increasing the unpaired electron density a t C4 and C6. Acknowledgment. We thank Kermit Mercer for invaluable technical assistance. This investigation was supported by PHS Grant 2-R37-CA32546, awarded by the National Cancer Institute, DHHS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. References and Notes ( 1 ) Goodhead, D.T.; Munson, R. J.;Thacker, J.;Cox,R. Int. J . Radial. Biol. 1980, 37, 135. (2) Ward, J . F.; Webb, C. F.; Limoli, C. L.; Milligan, J . R. In Ionizing Radiation Damage to DNA: Molecular Aspects; Wiley-Liss, Inc: New York, 1990; p 43. (3) Nabben, F. J.; Karman, J . P.; Loman, H. Int. J . Radial. Biol. 1982, 42, 23. (4) Rackovsky. S.;Bernhard, W. A . J . Phys. Chem. 1989, 93, 5006. (5) Hiittermann, J.; Voit, K.; Oloff, H. Faraday Discuss. Chem. Soc. 1984. 78. 135. (6) Sevilla, M. D.; Becker, D.; Yan, M.; Summerfield, S. R. J . Phys. Chem. 1991, 95, 3410. (7) Symons, M. C . R. J . Chem. Soc., Faraday Trans. I 1987, 83, I . ( 8 ) Bernhard, W . A. I n The Early Effecfs of Radiation on DNA; Fielden, E. M., O'Neill, P., Eds.; NATOASj-Series H 54; Springer-Verlag: Berlin. 1991: D 141. (9) Spailktta, R.; Bernhard, W . A . Radial. Res. 1992, 130, 7. (IO) Sevilla, M. D.; Failor, R.; Clark, C.; Holroyd, R. A,; Pettei, M. J . Phys. Chem. 1976, 80, 353. ( 1 1) AI-Kazwini. A. T.;O'Neill, P.;Adams,G. E.; Fielden, E. M . Radial. Res'. 1990, 121, 149. Charlton, D. E. Radial. Res. 1991, 125, 346. AIKazwini, A. T.; O'Neill. P.; Papworth, D.;Adams, G. E.; Fielden. E. M. Radiat. Res. 1991, 125. 348. (12) Cullis, P. M.; McClymont, J . D.;Symons, M. C . R. J . Chem. SOC., Faraday Trans. 1990. 86, 59 I , (13) van Lith, D.; Warman, J . M.; de Haas, M. P.; Hummel. A . J . Chem. SOC.,Faraday Trans. I 1986, 82, 2933. (14) Yan, M.; B'ecker. D.; Summerfield, S.; Renke, P.; Sevilla, M. D. J . Phys. Chem. 1992, 96. 1983. (15) Colson, A,; Besler, B.; Close, D. M.; Sevilla, M. D.J . Phys. Chem. 1992, 96, 661. (16) Telo, J . P.; Steenken, S.; Candeias, L. P. J . Am. Chem. SOC.1992, 114, 4701. (17) Steenken, S. Free Radical Res. Commun. 1992, 16, 349. (18) Madden, K. P.; Bernhard, W. A . J . Phys. Chem. 1979, 83, 2643. (19) Kar. L.; Bernhard, W. A. Radial. Res. 1983, 93, 232. (20) Barnes, J . ; Bernhard, W. A.; Mercer, K. R. Radial. Res. 1991, 126, 104.
(21) Mercer, K. R.; Bernhard, W . A . J . Magn. Reson. 1987, 74. 66. (22) Hiittermann, J.; Ohlmann, J.; Schaefer, A,; Gatzweiler, W. Int. J . Radial. Biol. 1991, 59, 1297.
3408 The Journal of Physical Chemistry, Vol. 97, No. 13, 1993 (23) Podmore, I. D.; Malone. M. E.; Symons, M. C. R.; Cullis. P. M.; Dalgarno, B. G. J. Chem. Soc.. Faraday Trans. 1991,87, 3647. (24) Plaush, A. C.; Sharp, R. R. J. Am. Chem. Soc. 1976. 98, 7973. (25) Walmsley, J. A.; Sagan, B. L. Biopolymers 1986, 25, 2149. (26) Sagarik, K . P.; Rode. B. M. fnorg. Chim. Acta 1983. 76. L209. Anwander. E. H. S.; Probst, M. M.; Rode, B. M. Biopolymers 1990,29,757. (27) The role of the anion, which has changed from CI to F , is not as obvious. It has been suggested that F may form a larger number of weaker hydrogen bonds that CI does, so that if the anion in the LiCl solution is partly responsible for the protonation shift to the amino group, the anion in the CsF solution should exert a smaller influence. For a discussion of the hydrogen bonding propertiesofanionsin aqueoussolutions, see: Strauss, I. M.;Symons, M. C. R. J. Chem. Soc., Faraday Trans I 1977, 73, 1796. (28) Symons, M. C. R. J. Chem. Soc., Perkin Trans. 2 1973, 791. (29) Sagstuen, E.; Hole, E. 0.; Nelson, W. H.; Close, D. M. J . Phys. Chem., in press. (30) Herak, J. N.; Lenard, D. R.; McDowelt, C. A. J . Magn. Reson. 1977,
26, 189. (31) Nakano, N. 1.; Igarashi, S. J . Biochemistry 1970, 9, 577
Barnes and Bernhard (32) Sanger, W. Principles of Nucleic Acid Strucrure; Springer-Verlag: New York, 1984; Chapter 6 and Chapter 13. (33) Wertz. J. E.; Bolton, J. R. Electron Spin Resonance: Elementary Theoryand Practical Application: McGraw-Hill: New York, 1972; Chapter 6. (34) Kyogoku. Y .; Lord. R. C.; Rich, A. J. Am. Chem. Soc. 1967.89.496. (35) In the X-irradiated high-salt glasses at 4 K, the trapped electron
signal isverypower broadenedand doesnotsignificantlyalterthe EPRspectra of the reduced bases. However, in the alcohol glasses, a 0.4-mTsinglet, whose height is comparable to the reduced base spectra, appears before photobleaching. Since the reduced adenine singlet spectrum is much sharper at g = 2.0023 than the reduced cytosine doublet, a small error due to the limited resolution in subtracting digitized spectra can lead to the appearance of a slight inflection point. (36) Nelson, W. H.;Sagstuen, E.;Hole, E. O.;Close, D.M.Radio?. Res., in press. (37) Candeias, L. P.; Steenken, S.J. Phys. Chem. 1992, 96,4701. (38) Weber, H. P.; Craven, B. M.Acta Crystallogr. 1990. 646, 532.
