Effect of base stacking on the acid-base properties of the adenine cation radical [A?+] in solution: ESR and DFT studies

Article abstract of DOI:10.1021/ja802122s

In this study, the acid-base properties of the adenine cation radical are investigated by means of experiment and theory. Adenine cation radical (A?⁺) is produced by one-electron oxidation of dAdo and of the stacked DNA-oligomer (dA)₆ by Cl₂?^- in aqueous glass (7.5 M LiCl in H₂O and in D₂O) and investigated by ESR spectroscopy. Theoretical calculations and deuterium substitution at C8-H and N6-H in dAdo aid in our assignments of structure. We find the pK_a value of A?⁺ in this system to be ca. 8 at 150 K in seeming contradiction to the accepted value of ≤1 at ambient temperature. However, upon thermal annealing to ≥160 K, complete deprotonation of A?⁺ occurs in dAdo in these glassy systems even at pH ca. 3. A?⁺ found in (dA)₆ at 150 K also deprotonates on thermal annealing. The stability of A?⁺ at 150 K in these systems is attributed to charge derealization between stacked bases. Theoretical calculations at various levels (DFT B3LYP/6-31G*, MPWB95, and HF-MP2) predict binding energies for the adenine stacked dimer cation radical of 12 to 16 kcal/mol. Further DFT B3LYP/6-31G* calculations predict that, in aqueous solution, monomeric A?⁺ should deprotonate spontaneously (a predicted pK_a of ca. -0.3 for A?⁺). However, the charge resonance stabilized dimer AA?⁺ is predicted to result in a significant barrier to deprotonation and a calculated pK _a of ca. 7 for the AA?⁺ dimer which is 7 pH units higher than the monomer. These theoretical and experimental results suggest that A?⁺ isolated in solution and A?⁺ in adenine stacks have highly differing acid-base properties resulting from the stabilization induced by hole derealization within adenine stacks.

Full text of DOI:10.1021/ja802122s

Published on Web 07/09/2008
Effect of Base Stacking on the Acid-Base Properties of the
Adenine Cation Radical [A•+] in Solution: ESR and DFT
Studies
Amitava Adhikary, Anil Kumar, Deepti Khanduri, and Michael D. Sevilla*
Department of Chemistry, Oakland UniVersity, Rochester, Michigan 48309
Received March 21, 2008; E-mail: sevilla@oakland.edu
Abstract: In this study, the acid–base properties of the adenine cation radical are investigated by means
of experiment and theory. Adenine cation radical (A•+) is produced by one-electron oxidation of dAdo and
of the stacked DNA-oligomer (dA)6 by Cl2•- in aqueous glass (7.5 M LiCl in H2O and in D2O) and investigated
by ESR spectroscopy. Theoretical calculations and deuterium substitution at C8-H and N6-H in dAdo
aid in our assignments of structure. We find the pKa value of A•+ in this system to be ca. 8 at 150 K in
seeming contradiction to the accepted value of e1 at ambient temperature. However, upon thermal annealing
to g160 K, complete deprotonation of A•+ occurs in dAdo in these glassy systems even at pH ca. 3. A•+
found in (dA)6 at 150 K also deprotonates on thermal annealing. The stability of A•+ at 150 K in these
systems is attributed to charge delocalization between stacked bases. Theoretical calculations at various
levels (DFT B3LYP/6-31G*, MPWB95, and HF-MP2) predict binding energies for the adenine stacked dimer
cation radical of 12 to 16 kcal/mol. Further DFT B3LYP/6-31G* calculations predict that, in aqueous solution,
monomeric A•+ should deprotonate spontaneously (a predicted pKa of ca. -0.3 for A•+). However, the
charge resonance stabilized dimer AA•+ is predicted to result in a significant barrier to deprotonation and
a calculated pKa of ca. 7 for the AA•+ dimer which is 7 pH units higher than the monomer. These theoretical
and experimental results suggest that A•+ isolated in solution and A•+ in adenine stacks have highly differing
acid-base properties resulting from the stabilization induced by hole delocalization within adenine stacks.
1. Introduction
(N1-H) is reported as 9.6, while the corresponding pKa of the
guanine cation radical (G•+) in H2O at ambient temperature is
ca. 3.9.3,4,8 The site of deprotonation in G•+ had been somewhat
controversial as it is reported to be N1 in solution in pulse
radiolysis studies and at the amine group (N2-H) in solid
state.9a Our recent ESR studies employing isotopically (15N and
deuterium) substituted dGuo confirm that the site of deproto-
nation in G•+ is at N1 in aqueous solution, and theoretical
calculations9a confirm shifting of that locus of deprotonation
of G•+ from N2 in a low dielectric solid9b to N1 in an aqueous
environment.9a
Electron and hole transfer through DNA is of significance to
an increasing number of areas of research including radiation
damage, DNA damage and repair processes, DNA photonics,
and electronics.1,2 Of the variety of processes that affect excess
electron and hole transfer through DNA, proton transfer is
perhaps the most important.1–7 Intrabase-pair proton transfer
processes have been proposed to slow or stop hole and excess
electron transfer through the helix and thereby act as a crucial
restriction on such spin and charge transfer.3–7 Such proton
transfer processes will strongly depend on the pKa of the base
ion-radical involved.
One-electron oxidized adenine (adenine cation radical (A•+)
and its deprotonated form (A(-H)•)) has also been well studied
(i) employing pulse radiolysis and flash photolysis in aqueous
solution,3,4,8,10,11 (ii) in γ-irradiated aqueous (D2O) glassy
systems at low temperature,12 (iii) in X-ray irradiated single
crystals,7,13–16 and in (iv) theoretical treatments.17–19,20a,b,21
Steenken and co-workers produced A•+ from dAdo in aqueous
The pKa of a cation radical is invariably found to be lower
than its parent compound. For example, the pKa of guanine
(1) (a) Becker, D.; Adhikary, A.; Sevilla, M. D. In Charge Migration in
DNA Physics, Chemistry and Biology PerspectiVes; Chakraborty, T.,
Ed.; Springer-Verlag: Berlin, Heidelberg, 2007; pp 139-175. (b)
Wang, Q.; Fiebig, T. In Charge Migration in DNA Physics, Chemistry
and Biology PerspectiVes; Chakraborty, T., Ed.; Springer-Verlag:
Berlin, Heidelberg, 2007; pp 221-248..
(8) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1989, 111, 1094–
1099.
(2) Wagenknecht, H.-A. In Charge Transfer in DNA: From Mechanism
to Application; Wagenknecht, H.-A., Ed.; Wiley-VCH Verlag GmbH
& Co. KGaA: Weiheim, 2005; pp 1-26..
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B. 2006, 110, 24171–24180. (b) Hole, E. O.; Nelson, W. H.; Sagstuen,
E.; Close, D. M. Radiat. Res. 1992, 129, 119–138.
(3) Steenken, S. Chem. ReV. 1989, 89, 503–520.
(4) (a) Steenken, S. Free Radical Res. Commun. 1992, 16, 349–379. (b)
Steenken, S. Biol. Chem. 1997, 378, 1293–1297.
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(11) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1992, 114, 699–704.
(12) Adhikary, A.; Becker, D.; Collins, S.; Koppen, J.; Sevilla, M. D.
Nucleic Acids Res. 2006, 34, 1501–1511.
(5) Colson, A.-O.; Besler, B.; Close, D. M.; Sevilla, M. D. J. Phys. Chem.
1992, 96, 661–668.
(6) Bertran, J.; Oliva, A.; Rodriguez-Santiago, L.; Sodupe, M. J. Am.
Chem. Soc. 1998, 120, 8159–8167.
(13) Close, D. M.; Nelson, W. H. Radiat. Res. 1989, 117, 367–378.
(14) Close, D. M.; Nelson, W. H.; Sagstuen, E.; Hole, E. O. Radiat. Res.
1994, 137, 300–309.
(7) Nelson, W. H.; Sagstuen, E.; Hole, E. O.; Close, D. M. Radiat. Res.
1998, 149, 75–86.
(15) Kar, L.; Bernhard, W. A. Radiat. Res. 1983, 93, 232–253.
9
10282 J. AM. CHEM. SOC. 2008, 130, 10282–10292
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2008 American Chemical Society

Acid-Base Properties of Adenine Cation Radical [A•+]
A R T I C L E S
solution employing 248 nm laser photolysis (20 ns pulses), and
from their measurements between pH’s 0 and 6, they concluded
that the pKa of A•+ is e1.3,4 This value is much lower than the
pKa of the corresponding N6-H atom (g14 4) in neutral dAdo.
Thus, ∆pKa(A/A•+) is much larger than the corresponding
∆pKa(G/G•+). However, in pulse radiolysis studies of dinucleo-
side phosphates ApG and GpA, rapid migration of the hole from
one-electron oxidized adenine to G was proposed and the rate
of this hole migration was shown to decrease by a factor of ca.
3 from pH 2.3 to 7.22 From these results, it was suggested that
the pKa of A•+ (e1) might be “too low” for these dinucleoside
phosphate systems (see footnote 25 in ref 22). In view of the
work to be presented here, these earlier results are the first results
suggesting that base stacking may affect the pKa of A•+. In a
much earlier NMR spectral study (360 MHz) of one-electron-
oxidized adenine produced via laser CIDNP in 5′-AMP at
various pH’s, the pKa of A•+ in aqueous (D2O) solution is
reported as 4 ( 0.2.23 Theoretical calculations predict the pKa
of A•+ as ca. 220a and 3.2.20b It is interesting to note that adenine
has been suggested to play an important role of a hole carrier
in long A-T stretches,24–27 and such hole transfer can occur
over long A stacks if G does not interrupt the A sequence.28
From the very low pKa value reported by Steenken (e1) of A•+,
it would seem likely that A•+ would deprotonate rapidly and
this deprotonation would stop the hole from being transferred
further in these adenine tracts. This apparently does not occur,
and one plausible explanation of this long-range hole transfer
in these A-tracts could be that the rate of hole transfer in these
adenine tracts is faster than the rate of its deprotonation.
Recently, the rate of hole transfer in such adenine tracts was
experimentally determined (kHT ) 108 to 1010 s-1),25 and this
value is slower than the expected rate of deprotonation base
cation radicals.3,4 From this one would incorrectly predict
deprotonation would occur during hole transfer. Recent work
by Barton and co-workers suggests that the hole within these
adenine tracts is delocalized over several adenine bases.27
Delocalization of the hole in A-tracts would likely stabilize the
Figure 1. Schematic diagram showing the atom numbering scheme of the
adenine ring in the deoxyadenosine cation radical (A•+) and the syn
conformation (with respect to N-atom N1 in the ring) of the deprotonated
cation radical (A(-H)•) is shown.
hole toward deprotonation, although no theoretical or experi-
mental studies have treated this issue. From the above discussion
it is clear that the nature of A•+ and its acid base properties are
still open questions in DNA systems.
In this work, we present experimental and theoretical work
that elucidates the acid–base properties of A•+. Following our
previous works,1,9,12 we have produced A•+ by one-electron
oxidation of dAdo by Cl2•- in an aqueous glass (7.5 M LiCl in
H2O and in D2O at various pH’s and concentrations). A•+ was
also formed via one-electron oxidation of the DNA-oligomer
(dA)6 by Cl2•- in an aqueous glass (7.5 M LiCl in H2O). We
have employed deuterium substitution in dAdo (at C8-H and
N6-H) aided with theoretical calculations to determine the
hyperfine coupling constants and to confirm our assignments
of structure (Figure 1). Theoretical calculations are also
employed to gain an understanding of the factors that increase
the stability of A•+, and these calculations predict that base
stacking can stabilize the cation radical state and can have
profound effects on its prototropic equilibria. The experimental
and theoretical results reported in this work suggest that, in
constrast to G•+, the properties of A•+ in the isolated monomeric
state can not explain the behavior of A•+ in DNA where stacking
dramatically changes the properties of this cation radical.
(16) Nelson, W. H.; Sagstuen, E.; Hole, E. O.; Close, D. M. Radiat. Res.
1992, 131, 272–284.
2. Materials and Methods
(17) Chen, Y.; Close, D. M. Struct. Chem. 2002, 13, 203–209.
(18) Wetmore, S. D.; Boyd, R. J.; Eriksson, L. A. J. Phys. Chem. B 1998,
102, 10602–10614.
2.1. Compounds. Lithium chloride (99% anhydrous, SigmaUltra)
and 2′-deoxyadenosine (dAdo) monohydrate were purchased from
Sigma (St. Louis, MO). We had obtained potassium persulfate
(crystal) from Mallinckrodt, Inc. (Paris, KY). Deuteration at C-8
in the adenine moiety was carried out following the procedure of
Huang et al.29 using triethylamine (TEA) (Fischer Scientific, NJ)
and D2O (Deuterium oxide, 99.9 atom % D) (Aldrich, Sigma-
Aldrich, Inc., St. Louis, MO). The degree of deuteration (g98%)
was confirmed by 1D NMR signal integration in DMSO-D6/99.9
atom % D (Wilmad LabGlass, Buena, NJ). The DNA-oligomer
(dA)6 used in this study was obtained from IDT-Synthegen with
standard desalting (Coralville, IA).
(19) Hwang, C. T.; Stumpf, C. L.; Yu, Y.-Q.; Kentta¨maa, H. I. Int. J. Mass
Spectrom. 1999, 182/83, 253–259.
(20) (a) Baik, M.-H.; Silverman, J. S.; Yang, I. V.; Ropp, P. A.; Szalai,
V. S.; Thorp, H. H. J. Phys. Chem. B 2001, 105, 6437–6444. (b) Chen,
X.; Syrstad, E. A.; Nguyen, M. T.; Gerbaux, P.; Turecˇek, F. J. Phys.
Chem. A 2004, 108, 9283–9293. (c) Jang, Y. H.; Goddard III, W. A.;
Noyes, K. T.; Sowers, L. C.; Hwang, S.; Chung, D. S. Chem. Res.
Toxicol. 2002, 15, 1023–1035.
(21) Nam, S. H.; Park, S. H.; Ryu, S.; Song, J. K.; Park, S. M. Chem.
Phys. Lett. 2008, 450, 236–242.
(22) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1993, 115, 2437–
2440.
2.2. Sample Preparation. Following our work,9,12 solutions of
0.3 to 10 mg of dAdo, its C-8-deuterated derivative, and of (dA)6
(2 mg) were prepared in 1 mL of 7.5 M in H2O and in D2O in the
presence of 3 to 6 mg of K2S2O8 as the electron scavenger. We
performed several experiments with 15 M LiCl to assess the effect
of LiCl on the prototropic equilibrium observed. pH’s of these
solutions were adjusted by quick addition of adequate amounts (in
µL) of 1 M NaOH or 1 M HCl in H2O or D2O under ice-cooled
conditions. pH papers were used for pH measurements of these
solutions. Due to this and also owing to the high ionic strength of
these solutions, we consider these pH values as approximations.9,12
(23) Scheek, R. M.; Stob, S.; Schleich, T.; Alma, N. C. M.; Hilbers, C. W.;
Kaptain, R. J. Am. Chem. Soc. 1981, 103, 5930–5932.
(24) Giese, B.; Amaudrut, J.; Ko¨hler, A.- K.; Spormann, M.; Wessely, S.
Nature 2001, 412, 318–320.
(25) (a) Kawai, K.; Majima, T. In Charge Transfer in DNA: From
Mechanism to Application; Wagenknecht, H.-A., Ed.; Wiley-VCH
Verlag GmbH & Co. KGaA: Weiheim, 2005; pp 117-151. (b) Lewis,
F. D.; Daublain, P.; Zhang, L.; Cohen, B.; Vura-Weis, J.; Wasielewski,
M. R.; Shafirovich, V.; Wang, Q.; Raytchev, M.; Fiebig, T. J. Phys.
Chem. B. 2008, 112, 3838–3843.
(26) Augustyn, K. E.; Genereux, J. C.; Barton, J. K. Angew. Chem., Int.
Ed. 2007, 46, 5731–5733.
(27) Shao, F.; O’Neill, M. A.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 17914–17919.
(28) Joy, A.; Ghosh, A. K.; Schuster, G. B. J. Am. Chem. Soc. 2006, 128,
5346–5347.
(29) Huang, X.; Yu, P.; LeProust, E.; Gao, X. Nucleic Acids Res. 1997,
25, 4758–4763.
9
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A R T I C L E S
Adhikary et al.
Figure 2. ESR spectra of one-electron oxidized adenine in dAdo or in 8-D-dAdo (3 mg/mL) produced by Cl2•- oxidation at 148 K (recorded at 77 K) in
7.5 M LiCl glass in H2O or in D2O in the presence of K2S2O8 at (A) native pH or pD (ca. 5) and at (B) pH or pD ca. 12. The three reference markers in
this figure and in all the other figures of this report showing ESR spectra are Fremy’s salt resonances with the central marker is at g ) 2.0056, and each of
the three markers is separated from one another by 13.09 G.
Deoxygenation of these solutions was carried out by bubbling
thoroughly with nitrogen followed by drawing the solutions into 4
mm Suprasil quartz tubes (cat. no. 734-PQ-8, WILMAD Glass Co.,
Inc., Buena, NJ). The transparent glassy samples were then prepared
by rapidly cooling the tubes containing these solutions to 77 K.
The 7.5 to 15 M LiCl used in this work are not crystalline solids
but glassy homogeneous supercooled liquids that on annealing
soften to allow for molecular migration and solution phase
chemistry. All these glassy samples were then stored in Teflon
containers at 77 K in the dark.9,12
2.3. γ-Irradiation and Storage of Irradiated Samples. γ-Ir-
radiation of the glassy samples of dAdo, of C-8-deuterated-dAdo
(i.e., 8-D-dAdo), and of (dA)6 were carried out under liquid nitrogen
using a model 109-GR9 irradiator, containing a shielded60Co source
as per our earlier work9,12 with an absorbed dose of 2.5 kGy (for
the nucleosides) and of 4.5 kGy (for the oligomer) at 77 K. All the
irradiated samples were also stored at 77 K in the dark.
2.6. Simulation of ESR Spectra. We have performed the
anisotropic simulations of the experimental ESR spectra using
SimFonia and WIN-EPR (Bruker) programs. The apparent g values
of A•+ and A(-H)• were determined from the field position of the
central component of their spectra (see Figures 2 and 3). Using
these g values, we have obtained the values of g| by comparing
the fits of the simulated spectra of A•+ and A(-H)• with the
corresponding experimentally obtained spectra of A•+ and A(-H)•
in 8-D-dAdo in D2O glass (see Figure 3). Theory predicts the major
axis of the nitrogen coupling (Azz) is collinear with the intermediate
proton couplings (Azz). This is expected since the pz-orbital on the
C8 atom should be coaxial with the π-orbitals on the ring, and the
optimized structures of A•+ and A(-H)• obtained via theoretical
calculations (see Supporting Information Figure S7) are only slightly
nonplanar. A•+ has 4 proton couplings (2 from the exocyclic
N-atom, and 1 each from the C2 and C8 atoms), and A(-H)• has
3 proton couplings (1 with exocyclic N-atom and 1 each from C2
and C8). These proton coupling tensors all have the z-axis colinear,
but the x and y axes are not. Hence, the calculated direction cosines,
L (see Supporting Information Table T1), were employed to rotate
the diagonalized A tensors to a common axis via (L-1A2L)1/2. The
experimental spectra for A•+ obtained either from dAdo or from
8-D-dAdo (see Figures 2 and 3) do not have any observable
resolution, and hence, we have used only the theoretically obtained
values for its simulation. On the other hand, the experimental spectra
of A(-H)• have observable resolutions both at the center and at
the wings (see Figures 2 and 3) from the amine hydrogen couplings
and the C-8 hydrogen coupling, as well as the nitrogen couplings.
By selective deuteration, we observe changes in spectral components
for A(-H)• which allows the estimation of the proton hyperfine
couplings. In the C-8 deuterated A(-H)• in D2O only the poorly
resolved nitrogen couplings are observed. Therefore, for A(-H)•
we have adjusted the theoretically obtained HFCC values reported
in the Table 1 to fit the experiment starting from the theoretically
predicted values (see Table 1 footnote d for these adjusted values
and the Supporting Information Table T1 for the original theoretical
predictions for all cases considered).
2.4. Formation of One-Electron Oxidized Adenine in dAdo
and (dA)6 by Thermal Annealing of Samples. One-electron
oxidized adenine species were produced by annealing γ-irradiated
glassy samples in a variable temperature assembly (Air Products)
in the dark via cooled nitrogen gas. The glassy samples were
annealed for 12-20 min at 148 to 152 K where the glass softens
sufficiently to allow for molecular migration which leads to the
disappearance of Cl2•- with the concomitant formation of one-
electron oxidized adenine (see Supporting Information Figure S1)
as described in our earlier work.12 After γ-irradiation at 77 K and
annealing, these glassy samples were immediately immersed and
stored in liquid nitrogen (77 K). To observe deprotonation, samples
were annealed further to slightly higher temperatures (ca. 160, 165
K) for 5-12 min and were stored immediately at 77 K in the dark
for ESR studies. As in our previous works,1,9,12 formation of sugar
radicals was not observed in these samples during annealing as
long as they were kept in the dark at all times.
2.5. Electron Spin Resonance. For recording ESR spectra of
these samples, we have used a Varian Century Series ESR
spectrometer operating at 9.2 GHz with an E-4531 dual cavity, 9-in.
magnet and with a 200 mW klystron.9,12 ESR spectra were recorded
at 77 K and at the microwave power range 25-40 dB. We have
used Fremy’s salt (with g ) 2.0056, AN ) 13.09 G) for field
calibration (shown as reference marks in all figures with ESR
spectra).9,12 As performed in our earlier works,9,12 a small singlet
“spike” from irradiated quartz at g ) 2.0006 was removed via
subtraction from the recorded spectra before analyses.
2.7. Theoretical (DFT) Calculations. The geometries of all
species described below were fully optimized at the B3LYP/6-31G*
level of theory using the Gaussian 03 suite of programs.30a We
have found optimized ground-state geometries for (i) deoxyad-
(30) (a) Frisch, M. J. et al. Gaussian03, revision B.04; Gaussian, Inc.:
Pittsburgh, PA, 2003 (for complete reference, see Supporting Informa-
tion). (b) Boys, S. F.; Bernardi, F. Mol. Phys. 1970, 19, 553.
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10284 J. AM. CHEM. SOC. VOL. 130, NO. 31, 2008

Acid-Base Properties of Adenine Cation Radical [A•+]
A R T I C L E S
Figure 3. ESR spectra for one-electron oxidized dAdo or 8-D-dAdo (3 mg/mL) at various pH’s in 7.5 M LiCl glasses in H2O (A-D) (black) and in D2O
(E-H) (black). The HFCC values and g-values used for the simulated spectra (red) are mentioned in Table 1. The spectra of A•+ and A(-H)• were recorded
at 77 K after annealing to 148 and 152 K, respectively. These results show that, up to 150 K, a one-electron oxidized adenine moiety either in dAdo or in
8-D-dAdo remains as A•+ in the pH range 3-7 and as A(-H)• at and beyond pH 9 either in H2O or in D2O glasses.
Table 1. B3LYP/6-31G* Calculated Hyperfine Coupling Constants (HFCCs) and Spin Densities at Different Atoms in A•+ and A(-H)•
hyperfine couplings (Gauss) ATot ) Aiso + Aii
atomic spin
densities
species
atom no.
Aiso
Azz
Ayy
Axx
a,b
A•+ (monomer)
N6
N7
N3
0.2499
0.1036
0.1525
0.1861
-
4.59
1.83
2.60
-5.74
-6.78
-6.69
-3.21
8.77
7.67
3.45
4.55
-0.74
-1.34
-1.49
-0.75
17.21
4.7
-3.8
-1.67
-2.25
3.697
6.38
5.85
2.51
-8.49
-2.30
-3.37
2.55
-3.88
-1.78
-2.297
-2.95
-5.04
-4.36
-1.77
-8.73
-2.39
-3.43
-1.74
gzz ) 2.0021
gyy ) 2.0041
gxx ) 2.0041
C8sHc
N6sHc
N6sHc
C2sHc
N6
line width (3.0, 2.5, 2.5 (D2O)) G,
and 4.0, 4.0, 4.0 (H2O)) G
-
0.0893
0.6176
0.1749
0.2380
0.1431
A(-H)• (monomer)a,b,c,d,e,f
gzz ) 2.0021
N1
2.51
4.15
-4.29
gyy ) 2.0045
N3
6.8
-0.32
gxx ) 2.0045
C8sHc
line widthd
N6sHc
-
-13.97
-10.36
13.47
-2.38
a For A•+ (monomer) and A(-H)•, optimization and HFCC values were calculated using DFT (B3LYP) with a 6-31G** basis set in the gas phase.
HFCC values obtained using 6-31G** and 6-31 G* basis sets were found to be very similar. b Direction cosines are given in the Table T1 in the
Supporting Information. c The spin density of the atom with which the H-atom is bonded, e.g., spin density on the C-atom in C8sH. d For A(-H)•, the
adjusted HFCC values (sum total of isotropic and anisotropic HFCC values) used to fit the experimental spectra in Figure 3 are N6 (21, 0.5, 0.5) G, N1
(5, 0.25, 0.25) G, N7 (9, 0.7, 0.7) G, C8-H (-5, -2.5, -7.5) G and N6-H (-20, 0.5, -11) G. e (4.5, 5.0, 6.0) G For A(-H)• in 8-D-dAdo in D2O
glass, (5.5, 6.0, 5.8) G for A(-H)• in dAdo in D2O glass; (4.2, 4.2, 4.2) for A(-H)• in 8-D-dAdo in H2O glass and (6.5, 4.2, 4.2) is for A(-H)• in
dAdo. f Calculations performed in the gas phase of A(-H)• (monomer) and AA(-H)• in the stacked conformation gave very similar HFCC values.
enosine cation radical (A•+) and (ii) syn- and anti-conformations
(with respect to the nitrogen atom, N1, in the ring) of the amine
deprotonated species, A(-H)• (see Figure 1 for atom numbering
of the adenine ring). The adenine dimer cation radical, i.e.,
(AB-AB)•+, was treated using the adenine base only. This dimer
cation radical was considered in two conformations: (i) in a planar
hydrogen-bonded conformation (AB-AB)p•+ and (ii) in the parallel
stacked conformation (AB-AB)s•+. The starting geometry for
optimization of the stacked conformation was generated by placing
the two adenine rings symmetrically along the axis normal to the
adenine ring plane and separated by 3.4 Å. Further, to consider the
effect of solvent surrounding the deoxyadenosine cation radical
(A•+), the geometry of A•+ was fully optimized in the presence of
seven water molecules. In addition, the base stacked dimer cation
radical ((AB-AB)s•+) was fully optimized in the presence of 18
water molecules. The polarized continuum model (PCM) using the
integral equation formalism (IEF) was used to take into account
the effect of the bulk solvent only for calculating the pKa of the
adenine base cation radical in the monomer, AB•+, and in the
stacked dimer (AB-AB)s•+ conformation (see Supporting Informa-
tion Tables T2 and T3). We note that theoretical calculations of
pKa values are very sensitive to the level of calculation; however,
Thorp and co-workers20a and Goddard and co-workers20c have
found the that B3LYP functional gives results which are in good
agreement with experiment. The hyperfine coupling constants
(HFCCs) of these adenine radicals were also calculated at the same
level of theory and basis set. Recently, the use of the B3LYP
functional and 6-31G* basis set was found to predict HFCCs in
goodagreementwithexperimentalvaluesinavarietyofmolecules.9a,31
Molecular structures were drawn using the JMOL molecular
modeling program32 while spin densities were plotted using the
GaussView33 molecular modeling software.
The basis set superposition error (BSSE) corrected stabilization
or binding energy of the adenine cation radical dimer is calculated
as follows:
9
J. AM. CHEM. SOC. VOL. 130, NO. 31, 2008 10285

A R T I C L E S
∆E ) Edimer-Eneutral-Ecation radical + BSSE
Adhikary et al.
using the apparent g value (2.0041) and the theoretically
calculated HFCC values (see Table 1) for A•+ in dAdo
(monomer, gas phase) and also for A•+ in 8-D-dAdo are shown
and have not been adjusted to fit the experiment. While the
simulated spectra for A•+ in D2O glasses are reasonable fits
with the corresponding experimentally obtained spectra (see
Figure 3E and 3F), they are somewhat broader in the corre-
sponding H2O spectra (see Figure 3A and 3B). Thus, from
Figure 3E and 3F, we conclude that the narrowing of the line
width upon C8-H deuteration is well predicted by theory.
However, there is a lack of resolution of individual components
which does not allow for further experimental assignment of
couplings. We find that the simulations are broader than the
experimental spectra (see Figure 3A and 3B). We suggest that,
for A•+, the exocyclic N-H HFCCs (see Table 1) are predicted
to be somewhat too large in H2O. Theoretical calculations for
stacked AA•+ suggest that owing to stacking induced charge
delocalization over both bases, the HFCC values of the exocyclic
N-H decrease, compared to their corresponding values pre-
dicted for the monomer cation radical (Vide infra, and see also
Supporting Information Figure S2, and Table T1), and this may
be a possible reason for the marginal fit.
(1)
where ∆E is the BSSE corrected stabilization or binding energy,
Edimer is the total energy of the adenine dimer cation radical, Eneutral
is the total energy of the neutral adenine (monomer), and Ecation
radical is the total energy of the adenine cation radical (monomer),
respectively. The BSSE correction was performed using the standard
Boys-Bernardi counterpoise correction scheme30b as implemented
in Gaussian03.30a To calculate the stabilization energy of the
adenine dimer cation radical, we have also employed the MP2 and
MPWB95 functional apart from B3LYP as implemented in
Gaussian03.30a Unless otherwise stated, all the energy values
mentioned in the text are BSSE corrected.
3. Results and Discussion
3.1. ESR Studies. 3.1.1. ESR Spectra of One-Electron
Oxidized Adenine at ca. pH 5 and at ca. pH 12 in H2O and
in D2O glasses at 150 K. In Figure 2, we present the ESR spectra
of one-electron oxidized dAdo and 8-D-dAdo at the native pH
(ca. 5) and also at ca. pH 12 of 7.5 M LiCl either in H2O or in
D2O at 148 K. At pH 5 the cation radical, A•+, is found,
whereas, at pH 12, the deprotonated radical, A(-H)•, is found.
The various spectra in Figure 2A and 2B show the results of
deuterium substitution at various sites and help to assign
hyperfine couplings. Exocyclic amine hydrogens in dAdo (or
in Ado) are exchangeable in D2O, and deuterons show hyperfine
couplings 1/6.5 of that of protons in the same environment.9a
Thus hydrogen couplings that are observed in H2O are lost in
D2O (see Figure 2). This, along with larger dipolar interactions
from the solvent in H2O vs D2O, explains the increase in the
spectral width in H2O compared to that in D2O. Isotopic
substitution of the C8-H atom in dAdo by deuterium is found
to narrow the line width by ca. 4-5 G either for A•+ (see Figure
2A) or for A(-H)• (see Figure 2B). The constant field position
of the central component (apparent g value) for each of the
conditions in Figure 2A is consistent with A•+ maintaining its
molecular and electronic structure on isotopic substitution. A
similar argument holds good for A(-H)• found in H2O or in
D2O for dAdo and for 8-D-Ado at pH 12 (see Figure 2B). Note
that the ESR spectrum of A•+ shows a clear and observable
difference in the field position of the central component
(apparent g value) when compared to that in the spectrum of
its deprotonated form A(-H)• (see Figure 3). The apparent g
value (2.0041) of A•+ is lower than the corresponding value
(2.0045) of apparent g of A(-H)•, and these values have been
used for simulation of ESR spectra of these radicals (see Table
1).
We have presented the ESR spectra of A(-H)• at pH or pD
12 in Figure 3C, 3D and 3G, 3H formed after annealing at 152
K, via one-electron oxidation by Cl2•- in the glassy (7.5 M
LiCl) samples of dAdo and 8-D-dAdo either in H2O or in D2O
along with the corresponding spectral simulations in red. For
A(-H)•, identical ESR spectra are found at pH’s (pD’s) from
9 to 12. Simulations were performed employing apparent g
and g| values of 2.0045 and 20025 as well as HFCCs reported
in Table 1 for A(-H)•. These simulations (shown in red color)
match the experimental spectra (3C, 3D) and (3G, 3H) quite
well. In these cases both the amine hydrogen couplings, the
C-8 hydrogen coupling, and the nitrogen couplings are observ-
able in the spectra. Hence, the corresponding calculated HFCC
values reported in the Table 1 were adjusted to fit the
experiment, and these are reported in footnote d of this table.
In going from D2O to H2O glasses (compare Figure 3G vs 3C)
we note the hyperfine couplings of the exchangeable N6-H
proton coupling become clearly distinguishable. In addition, we
note that the line width and spectral shape changes on
deuteration at C8 (compare 3C with 3D and 3G with 3H) are
well predicted by the simulations. The adjusted couplings
reported in Table 1 for A(-H)• are expected to be good
estimates of the major coupling values. We also note that
theoretical calculations performed for A(-H)• (monomer) and
AA(-H)• in the stacked conformation showed little effect of
stacking and gave very similar HFCC values (see footnote h in
Supporting Information Table T1).
In the pH (or pD) range 3-7, the ESR spectrum for A•+ is
found for dAdo and for 8-D-dAdo, whereas, in the pH (or pD)
range 9-12, we find the ESR spectrum for A(-H)•. We have
found at pH 8 at 150 K the ESR spectrum of A•+ and A(-H)•
found in equal amounts. They are presumed to be in equilibrium
at 150 K. This ESR spectrum and its analyses are now shown
in Supporting Information Figure S3. This clearly indicates the
pKa of A•+ in dAdo in the H2O glassy samples is ca. 8 (see
Figure 4A) at 150 K. In D2O glassy samples at 155 K, we find
the pKa of A•+ in dAdo is ca. 8.5. Moreover, we find no
temperature dependence in the amounts of A•+ and A(-H)• at
pH 7.5 from 77 to 150 K (see Supporting Information Figure
S3).
3.1.2. ESR Spectra of One-Electron Oxidized Adenine at
Various pH Ranges, e.g., 3-7 and 9-12 in H2O and in D2O
Glasses. The ESR spectra of A•+ formed after annealing at 148
K in the dark, via one-electron oxidation by Cl2•- in the glassy
(7.5 M LiCl) samples of dAdo and 8-D-dAdo either in H2O or
in D2O and at pH (pD) 5 are shown in Figure 3A, 3B and 3E,
3F along with spectral simulations in red. The spectra shown
in Figure 3A, 3B and 3E, 3F remain identical in the pH or pD
range 3-7. The spectral shape, total hyperfine splitting, and
the field position of the central component of spectrum 3E match
very nicely with that already reported for A•+ in the literature
(Figure 7A in Sevilla et al.34a). Simulation (shown in red color)
(31) Hermosilla, L.; Calle, P.; Garc´ıa, de la Vega, J. M.; Sieiro, C. J. Phys.
Chem. A 2006, 110, 13600–13608.
(32) http://jmol.sourceforge.net. Jmol development team, An Open-Science
Project 2004.
(33) GaussView; Gaussian, Inc.: Pittsburgh, PA, 2003.
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Acid-Base Properties of Adenine Cation Radical [A•+]
A R T I C L E S
literature, and their values are -4.2 kJ mol-1, -27.2 kJ mol-1
,
and -75.2 J K-1 mol-1, respectively.34b,cSince concentration
of dAdo in our system (3 mg/mL and above) falls in this range
where dAdo is reported to associate and as the nature of our
system (glassy at low temperature (ca. 148 to 165 K)) promotes
stacking in dAdo, we propose that the adenine cation radical
exists primarily in the stacked form (i.e., as a dimer) in our
system. This explains its high pKa value (ca. 8) at 150 K.
Progressive warming to higher temperatures allows for molec-
ular migration and likely disrupts the dimer cation radical to
the monomer cation radical with it is low pKa value, thereby
leading to its immediate deprotonation. This would explain our
observation that A•+ is completely converted to A(-H)• upon
annealing to ca. 160 K in H2O glass (see Figure 4B).
We find that, with increasing concentration of dAdo, the
extent of A•+ formed at 152 K increases (see Supporting
Information Figure S6B). Thus, the stacking phenomenon tends
to stabilize the cation radical state of the one-electron oxidized
adenine moiety more with respect to its deprotonated state. We
have also observed a shift of field position of the central
component (apparent g value) of one-electron oxidized adenine
formed by annealing at 152 K as a function of dAdo concentra-
tion (see Supporting Information Figure S6A). This change in
the apparent g value is likely associated with increasing
association of the adenines.
Figure 4. (A) The schematic diagram shows that A•+ is found for pH’s
up to 7 and A(-H)• is found at pH’s of 9 and above at 150 K in 7.5 M
LiCl H2O or D2O glasses. At pH 8, equal amounts of A•+ and A(-H)• are
found; the pKa of A•+ is therefore ca. 8 at 150 K in these systems. In D2O
glassy samples, the corresponding pKa of A•+ is ca. 8.5. (B) The prototropic
equilibrium of A•+ is found at 150 K. However, annealing to a slightly
higher temperature of ca. 160 K and above allows for molecular migration,
and deprotonation is found at all pH’s investigated, i.e., 3 to 12, in H2O
glasses. In D2O glasses, which soften at slightly higher temperatures,
deprotonation of A•+ is nearly complete at 165 K.
3.1.5. A•+ and A(-H)• Formed in the DNA-Oligomer
(dA)6: Effect of Base Stacking. In this work we have used (dA)6
to probe the influence of stacking interaction on the ESR spectral
characteristics of A•+ and A(-H)• with respect to the monomer
(i.e., dAdo). CD and NMR studies show that oligomers (dA)n
in aqueous solution at 273 K are in the stacked form.34d The
dimer, (dA)2, is found to be 90% in the stacked form with greater
degrees of stacking observed as n increases. Therefore, in our
experimental conditions which promote stacking (frozen aqueous
solution at 150 K), the oligomer (dA)6 should exist entirely in
the stacked form. In Figure 5D, we present our results of (dA)6
at 148 K and after annealing to 160 K. The spectra of A•+ and
A(-H)• formed in (dA)6 match closely with those for the
monomer (compare spectrum 5D with 5A and spectrum 5E with
5C). This further strengthens our conclusion that the cation
radical exists in the stacked form even in dAdo.
3.1.3. Annealing Studies of A•+ Formed from dAdo. Spectra
in Figure 5A to 5C show that, at pH 3, annealing A•+ in samples
of 3 mg/mL dAdo in 7.5 M LiCl/H2O to 160 K results in
complete deprotonation to form A(-H)•. Identical results are
found at pH 5 (see Supporting Information Figure S4). No
significant deprotonation of A•+ is observed after annealing at
148 K. However, after annealing to 152 K for 15 min, in the
pH range 3-7, the ESR spectrum of one-electron oxidized dAdo
in H2O glasses show about 25-30% deprotonation; i.e., the
spectral composition is 70-75% A•+ and 25-30% A(-H)•.
One question could be, what is the effect of the LiCl matrix
on the prototropic equilibrium of A•+? We have observed that
(i) one-electron oxidized adenine in dAdo gave nearly identical
spectra and pH dependence for A•+ and A(-H)• even at 15 M
LiCl and, (ii) even at 15 M LiCl, A•+ is converted to A(-H)•
upon annealing (see Supporting Information Figure S5). This
suggests that the LiCl matrix does not have a large effect on
the prototropic equilibrium of A•+ in dAdo.
As shown in Figure 4B, at 160 K, we find less conversion of
A•+ to A(-H)• in D2O glasses in comparison to H2O glasses.
For example, in D2O glasses ca. 30% of A•+ is converted to
A(-H)• in dAdo on annealing to 160 K with near complete
deprotonation at 165 K. This is attributed to the somewhat higher
softening point of the D2O glass in comparison to the H2O glass.
3.1.4. Effect of Stacking on A•+ and A(-H)• Formed from
dAdo in H2O Glasses. It is well established in the literature that,
in aqueous solution at ambient temperature, dAdo dimerizes in
a stacking geometry in the concentration range of millimolar
and above.34b,c,e,f The thermodynamic parameters (∆G°, ∆H°,
∆S°) of this dimerization have also been reported in the
3.1.6. Annealing Studies of A•+ Formed the DNA-Oligomer
(dA)6 in H2O Glasses. We find that A•+ in the DNA-oligomer
(dA)6 forms A(-H)• upon annealing to 160 K in the dark at
pH (ca. 5) in 7.5 M LiCl (H2O) glass (see spectra 5D and 5E).
The behavior of (dA)6 is identical to that of dAdo on
annealing to 160 K.
3.2. Theoretical Studies. 3.2.1. Deoxyadenosine Cation
Radical (A•+) Monomer Calculations. The B3LYP/6-31G(d)
optimized structures of the deoxyadenosine radical cation (A•+)
and deprotonated A•+ in syn- and anti-conformations (A(-H)•syn
and A(-H)•anti) with respect to the N1 atom of A•+ are shown
in Supporting Information Figure S7. For A•+ we found that
B3LYP/6-31G* calculated major hyperfine couplings (HFCCs)
are localized at N6, N7, N3, C8, H′, H′′, and C2 atoms,
(34) (a) Sevilla, M. D.; Becker, D.; Yan, M.; Summerfield, S. R. J. Phys.
Chem. 1991, 95, 3409–3415. (b) Ts’o, P. O. P. In Fine Structure of
Proteins and Nucleic Acids; Fasman,G. D.,; Tiemasheff,S. N., Eds.;
Marcel Dekker: New York, 1970; p 151. (c) Broom, A. D.; Schweizer,
M. P.; Ts’o, P. O. P. J. Am. Chem. Soc. 1967, 89, 3612–3622. (d)
Olsthoorn, C. S. M.; Bostelaar, L. J.; De Rooij, J. F. M.; Van Boom,
J. H.; Altona, C. Eur. J. Biochem. 1981, 115, 309–321. (e) Itahara,
T.; Imaizumi, K. J. Phys. Chem. B 2007, 111, 2025–2032. (f) Leonard,
N. J. Acc. Chem. Res. 1979, 12, 423–429.
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A R T I C L E S
Adhikary et al.
Figure 5. ESR spectra for one-electron oxidized dAdo (3 mg/mL) in pH 3 (A-C) and for one-electron oxidized DNA-oligomer (dA)6 (2 mg/mL) at the
native pH (ca. 5) (D, E) in 7.5 M LiCl glass in H2O (black). Spectra A and D are the spectrum of corresponding A•+ recorded at 77 K after its production
via annealing to 148 K in the dark. Spectra B, C at pH 3 and spectrum E at pH 5 show that, upon annealing at 160 K in the dark, A•+ is converted completely
to A(-H)•. The authentic spectrum of A(-H)• (red) obtained by one-electron oxidation of dAdo at pH 12 in H2O glass is superimposed on spectra B, C,
and E for comparison. All spectra were recorded at 77 K.
respectively (see Table 1). In the gas phase, the A(-H)•syn
conformation was more stable by ca. 1.04 kcal/mol than the
corresponding A(-H)•anti conformation as predicted by the
B3LYP/6-31G* method. The B3LYP/6-31G* calculated HFCCs
of the A(-H)•syn conformation are localized on atoms N6, N1,
N3, C8, and H′′, respectively (see Table 1).
In Figure 6a and b, we present the optimized geometries of
adenine cation radical dimer hydrogen bonded ((AB-AB)p•+)
in C1 symmetry and in a stacked conformation [(AB-AB)s•+]
in Cs symmetry (parallel orientation). Both calculations are for
the gas phase.
The B3LYP/6-31G* optimized structure of the deoxyadenos-
ine cation radical (A•+) in the presence of seven water molecules
(A•+ + 7H2O) is shown in Figure 6c. To reduce the compu-
tational cost, we placed water molecules only near the adenine
base. Based on the hydrogen-bond distances (see Figure 6c), it
is evident that the hydrogen bonds between a water molecule
and the H′ and H′′ hydrogens of the NH2 group of the adenine
base are quite strong. In the hydrated complex (A•+ + 7H2O),
we found that the N6-H′ bond is elongated by ∼0.1 Å as
compared to the N6-H′′ bond (see Figure 6c) or the corre-
sponding N-H bonds in A•+ in the gas phase (see Supporting
Information Figure S7). Leszczynski and co-workers recently
studied the hydration of adenine in the presence of 12, 13, 14,
and 16 water molecules using the B3LYP/6-31G(d) method.35
The locations of the water molecules in their study are nearly
identical to our work (see Figure 6c). In the optimized structure,
we found that the water molecules form hydrogen bonds with
the N1, H′, H′′, N3, H2, and N7 atoms of the adenine ring and
lie in the range ∼1.431 to 2.159 Å, respectively, and these values
are very similar to the corresponding values of Leszczynski and
co-workers.35
is shown in Figure 6b. To simplify the computational complex-
ity, we consider only the adenine base (AB) without the sugar
moiety in the dimer calculation. Generally, it is found that the
B3LYP method is not suitable for systems that are bound by
weak π-π stacking interaction energies which are dispersion-
like interactions.36 Since our stacked conformation (AB-AB)s
also involves such interactions, first we have checked the
reliability of the B3LYP method with the 6-31G* basis set and
optimized the structures of the benzene dimer in the neutral
state and also in the cation radical state as test cases. We note
that the benzene cation radical ((C6H6)n•+, n ) 2 to 6) has been
studied by several groups using DFT and these clusters are
stabilized owing to charge resonance interaction.37a–e In ac-
cordance with the existing literature, we found that the B3LYP
method fails to predict π-stacking interactions for the neutral
stacked conformation but predicts strong interactions for the
benzene dimer cation owing to charge resonance interactions.
Optimization using the B3LYP/6-31G* method shows that the
benzene rings in the cation radical are separated by ca. 3.51 Å
while in the neutral states the rings separate to a noninteracting
distance of ca. 6 Å, respectively. We find that the BSSE
corrected B3LYP/6-31G* calculated stabilization energy for the
benzene cation radical dimer is -17.65 kcal/mol. The BSSE
corrected MP2/6-31G*//B3LYP/6-31G* calculated (single point)
stabilization energy for the benzene cation radical in the dimer
conformation is found to be -17.56 kcal/mol. These values are
in close agreement with the experimentally measured binding
(36) Zhao, Y.; Truhlar, D. G. Acc. Chem. Res. 2008, 41, 157–167.
(37) (a) Pieniazek, P. A.; Krylov, A. I.; Bradforth, S. E. J. Chem. Phys.
2007, 127, 044317. (b) Rusyniak, M. J.; Ibrahim, Y. M.; Wright, D. L.;
Khanna, S. N.; El-Shall, M. S. J. Am. Chem. Soc. 2003, 125, 12001–
12013. (c) Itagaki, Y.; Benetis, N. P.; Kadam, R. M.; Lund, A. Phys.
Chem. Chem. Phys. 2000, 2, 2683–2689. (d) Todo, M.; Okamoto, K.;
Seki, S.; Tagawa, S. Chem. Phys. Lett. 2004, 399, 378–383. (e)
Okamoto, K.; Seki, S.; Tagawa, S. J. Phys. Chem. A 2006, 110, 8073–
8080. (f) Howarth, O. W.; Frenkel, G. K. J. Am. Chem. Soc. 1966,
88, 4514–4515. (g) Badger, B.; Brocklehurst, B. Nature 1968, 219,
263.
The B3LYP/6-31G* optimized structure of the adenine base
cation radical dimer in the stacked conformation (AB-AB)s•+
(35) Sukhanov, O. S.; Shishkin, O. V.; Gorb, L.; Podolyan, Y.; Leszczynski,
J. J. Phys. Chem. B 2003, 107, 2846–2852.
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Acid-Base Properties of Adenine Cation Radical [A•+]
A R T I C L E S
Figure 6. B3LYP/6-31G* optimized geometries of (a) the hydrogen bonded adenine cation radical dimer, (AB-AB)p•+, in C1 symmetry in the gas phase.
(b) Adenine dimer cation radical [(AB-AB)s•+] in a stacked parallel conformation in the gas phase. The geometry was optimized in Cs symmetry. (c)
Deoxyadenosine cation radical in the presence of seven water molecules (A•+ + 7H2O). The arrow on the elongated N6-H′ bond in this figure indicates
that the elongation of this N6-H′ bond leads to deprotonation in the surrounding water shell (shown in e). (d) Adenine dimer cation radical in stacked
conformation surrounded by 18 water molecules [(AB-AB)s•+ + 18H2O] (local minimum), and (e) hydronium ion formation from A•+ owing to deprotonation
of the structure in (c).
energy (17.6 kcal/mol) of the benzene radical cation dimer by
Rusynial et al.38 using mass-selected, ion mobility drift-cell
techniques. From these results it is clear that the π-π charge
resonance interactions are well predicted in stacked π-system
cation radical dimers but the far weaker π-π dispersion
interactions in a neutral system are poorly treated as expected.
To calculate the stabilization energy of the adenine cation
radical in the stacked conformation, we employed B3LYP as
well as MPWB95 and MP2 methods that better account for
dispersion interactions. In the present study, we considered two
different arrangements of the adenine ring in the stacked
conformations: (i) parallel conformation, in which two adenine
rings are placed parallel to each other, and (ii) antiparallel
conformation, in which one of the adenine ring plane was rotated
180° with respect to the other adenine ring plane. The geometries
in these two stacked conformations were fully optimized in their
neutral and cationic radical state using MP2, B3LYP, and
MPWB95 methods and the 6-31G* basis set (see Supporting
Information Figures S11 and S12). The BSSE corrected
stabilization energies (in kcal/mol) along with the interbase
distances (in angstroms) are presented in Table 2. From Table
2, we note that stabilization energies for the dimer cation radical
lie in the range -11.5 to -16.3 kcal/mol. In addition, for the
dimer cation, the antiparallel conformation was found to be only
slightly more stable (∼ -0.7 to 3 kcal/mol) than the parallel
conformation (see Table 2). However, in the neutral state, the
antiparallel conformation was found to be ∼ -5 to 6 kcal/mol
(38) Rusyniak, M.; Ibrahim, Y.; Alsharaeh, E.; Meot-Ner (Mautner) , M.;
El-Shall, M. S. J. Phys. Chem. A 2003, 107, 7656–7666.
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Adhikary et al.
Table 2. Stabilization Energies of Adenine Dimer Cation Radical in
distances than those found for the gas-phase stacked system
(see Figure 6b). We also found the Mulliken spin densities and
the HFCCs in the (AB-AB)s•+ + 18 H2O complex favor one
of the adenine rings (see Table T1 in the Supporting Informa-
tion). The spin density distribution for stacked (AB-AB)s•+ in
Various Stacked Formsa
Eint (kcal/mol)
interbase distance (Å) (BSSE corrected)
parallelb antiparallelb parallelb antiparallelb
method
(all full optimization)
symmetry
the gas phase and in the hydrated environment (AB-AB)s•+
+
Dimer Cation Radical
3.4-3.9
B3LYP/6-31G*
MP2/6-31G*
MPWB95/6-31G* Cs
Cs
Cs
–
-12.08
–
18 H2O are shown in Figure 7a and b.
2.9-3.4 2.9-3.2 -15.64 -16.32
From Figure 7, it is clearly evident that spin densities are
more localized (73% vs 27%) on one of the adenine rings in
the (AB-AB)s•+ + 18 H2O complex; however, it is fully
delocalized in the gas phase (AB-AB)s•+. It appears that the
surrounding water molecules adjust their structure on optimiza-
tion to more strongly interact with one of the adenine rings and
thereby tend to localize the spin density. The driving force for
the localization stems from the stronger hydrogen bonds formed
with the adenine ring with the greater charge (spin). Thus, a
partially charge-separated state such as (A•+-A) in the hydrated
environment is likely the minimum energy configuration. In
order to test whether the spin densities shown in Figure 7 are
artifacts of the B3LYP calculations, we also plotted the spin
densities using Hartree-Fock (HF) as well as BHandHLYP
methods and we have found very similar results (see Supporting
Information Figure S10). In a recent study for [dApdA]•+ by
Kumar and Sevilla,39a it has been observed that the spin densities
are delocalized on both of the adenine rings; however, the spin
densities were found to be localized on one of the adenine rings
for [[dApdA(-H)•]. Similar results were found by Parinello and
co-workers.39b We also note that the spin densities in the
benzene dimer cation radical are well established to be delo-
calized on both rings equally by EPR studies and theory.37c EPR
studies of the benzene dimer cation radical also shows that the
hyperfine coupling constants for the benzene dimer cation are
exactly one-half of those for the monomer as expected.37c This
phenomenon is found for other aromatics such as naphthalene
and anthracene.37f,g Thus, we conclude that this delocalization
of charge in the aromatic dimer cation radical is not an artifact
of the calculations and will apply to adenine as well.
3.2-3.5 3.1-3.4b -11.54 -14.76
MP2/6-31G*
C1 (skew)
2.9-3.5
-13.54
Neutral Dimer
MPWB95/6-31G* Cs
3.5-3.6 3.2-3.4
3.4-3.5
3.06
-0.91
-2.91
-5.8
MP2/6-31G*
MP2/6-31G*
Cs
C1
3.1-3.4
a Gas phase calculations. Energies calculated using eq 1. All
calculations are BSSE corrected. b The parallel conformation was
generated by placing the two adenine rings symmetrically along the axis
normal to the adenine ring plane. The starting geometry for antiparallel
conformation was obtained by rotating one of the two adenine rings in
the parallel conformation by 180° (see Supporting Information Figures
S11 and S12).
more stable than the parallel conformation (see Table 2). Thus,
in comparison to the neutral system where the two rings are
stabilized mainly due to dispersion, the radical cation systems
are held together very strongly in the stacked conformation due
to charge resonance interactions. In addition, the stabilization
energy -12.08 kcal/mol calculated using the B3LYP method
for the radical cation is close to the value -15.64 kcal/mol as
calculated by the MP2 method (see Table 2). Further, this
detailed analysis clearly shows the applicability of the B3LYP
method to study this class of systems.
From Table T1 (see Supporting Information), we found that,
in the stacked conformation (see Figure 6b), the Mulliken spin
densities are equally delocalized in both adenine rings (as
evidenced by the HFCC values) and, in each ring, the spin
densities are located mainly on atoms N6, N3, C8, H′, and H′′,
respectively.
The B3LYP/6-31G* optimized structure of hydrogen-bonded
(AB-AB)p•+ is shown in Figure 6a as well as in Figure S8 in
the Supporting Information. This optimized structure
((AB-AB)p•+) was obtained when the stacked (AB-AB)s•+
conformation was optimized in the C1 symmetry. In the
hydrogen-bonded dimer cation radical ((AB-AB)p•+), the two
adenine rings are twisted and hydrogen bonded with each other
having hydrogen bond distances 1.293 and 2.353 Å, respectively.
The BSSE corrected B3LYP/6-31G* calculated binding energy
of the hydrogen bonded dimer cation radical was found to be
18.98 kcal/mol. Recently, Nam et al.21 studied the adenine cation
radical dimer using the B3LYP/6-31+G(d,p) method. They
reported a planar hydrogen-bonded adenine dimer cation radical
having a binding energy of 1.08 eV (24.91 kcal/mol, without
BSSE correction). The difference in the binding (24.91 and
18.98 kcal/mol) results from slightly different structures and
lack of BSSE correction. The calculated spin distribution in
(AB-AB)p•+ is more localized on one of the adenine rings on
atoms N6, N3, and C8 (see Table T1 in the Supporting
Information).
Using the B3LYP/6-31G* method with the PCM model for
solvent interactions, we also calculated the pKa of the adenine
cation radical (A•+) and the stacked adenine cation radical
(AB-AB)s•+ and present the calculation details in Tables T2
and T3 in the Supporting Information. The theoretical calculation
of pKa of a molecule is very sensitive, and a small change in
the calculated free energy (G) can change the pKa values by
several units (orders of magnitude in Ka). In our case, we
calculated the pKa using the free energy of H+ in the gas phase
as -6.28 kcal/mol based on the Sackur-Tetrode equation.20a
Deprotonation from the NH2 group of AB•+ was considered,
and the calculated pKa values of AB•+ in anti- and syn-
conformations are -0.26 and -1.04 (see Supporting Information
Figure S9) while the corresponding values for (AB-AB)s•+ are
7.02 and 6.44, respectively. Based on the calculated pKa values,
the monomer, AB•+, is found to be more acidic than the dimer,
(AB-AB)s•+, by ca. 7 pKa units. In this context, we calculated
the potential energy surface (PES) for the deprotonation of the
NH2 group considering the optimized structures of A•+ + 7
H2O and (AB-AB)s•+ + 18 H2O shown in Figure 8. In
calculating the PES, the N6-H′ bond was elongated from its
equilibrium bond distance in 0.1 Å steps. At each fixed N6-H′
distance the geometry was fully optimized. For A•+ + 7 H2O,
To examine the effect of the surrounding water molecules
on the stacked conformation, we optimized the stacked adenine
cation radical in the presence of 18 water molecules using the
B3LYP/6-31G* method. The optimized local minimum structure
of (AB-AB)s•+ + 18 H2O is shown in Figure 6d. We found
that in the (AB-AB)s•+ + 18 H2O complex the two adenine
rings are stacked within ca. 3.45 to 3.62 Å. These are smaller
(39) (a) Kumar, A.; Sevilla, M. D. J. Phys. Chem. B 2006, 110, 24181–
24188. (b) Mantz, Y. A.; Gervasio, F. L.; Laino, T.; Parinello, M. J.
Phys. Chem. A 2007, 111, 105–112.
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10290 J. AM. CHEM. SOC. VOL. 130, NO. 31, 2008

Acid-Base Properties of Adenine Cation Radical [A•+]
A R T I C L E S
Figure 7. B3LYP/6-31G* calculated spin-density distribution shown in percentages in (a) the base stacked adenine dimer cation radical [(AB-AB)s•+] in
the gas phase and (b) the base stacked adenine dimer cation radical surrounded by 18 water molecules. On optimization, the water molecules adjust to favor
spin on one of the adenine rings in the [(AB-AB)s•+ + 18H2O] complex.
tions, the adenine cation radical (A•+) and its deprotonated
radical (A(-H)•) have been identified in glassy systems of dAdo
and (dA)6. The literature suggests that both the monomer dAdo
and the DNA-oligomer (dA)6 should have the adenine bases in
astackedconfigurationinaqueoussolutionsatlowtemperatures.34b,c,e,f
Our results suggest that this stacking stabilizes A•+ by partial
hole delocalization to adjacent adenines. We note that, in spite
of base stacking, an isolated A•+ is found in irradiated
adenosine · HCl hemihydrate single crystals16 and A(-H)• is
found in adenosine single crystals.13 However, in each case the
initial hole has undergone a deprotonation reaction from the
parent structure which allows for its localization on a single
base. Hole localization on the deprotonated base in base stacks
has been reported in our previously reported calculations for
adenine dinucleoside phosphate systems as well.39a These
Figure 8. Potential energy surface (PES) of N6-H′ bond dissociation of
the (a) A•+ + 7H2O cation radical and (b) (AB-AB)s•+ + 18H2O cation
radical in stacked conformation. The distances and energies are given in
angstroms (Å) and kcal/mol, respectively. See Figure 6 for structures.
calculations show that, in the A(-H)• stack, spin is localized
only on the A(-H) portion of the dinucleoside phosphate as
expected; however, the spin is delocalized in the AA stack in
the cation radical of the dinucleoside phosphate.39a In aqueous
solutions of Ado and dAdo at ambient temperatures and low
concentrations where no stacking occurs,34b,c pulse radiolysis
experiments find only A(-H)• as expected from the low pKa
of monomeric A•+.3,4,8,10,11
Table 3. Summary of the Experimental and Theoretically
Calculated Properties of A•+ and of A2•+
nature of
activation barrier
for N6-H′
BSSE corrected
binding energy
for (AB-AB)s•+
(kcal/mol)
est pKa
deprotonation
system
expt (150 K) theorya
theoryb
theory
(ii) pKa values and dimer cation stabilization (theory and
experiment): Experimentally we find that A•+ in dAdo in our
aqueous system has the pKa ca. 8 at 150 K, which deprotonates
upon thermal annealing at 160 K (see Figures 4 and 5).
However, theoretical calculations for the pKa (B3LYP/6-31G*)
predict the pKa of A•+ is ca. -0.3 and also predicts the pKa of
the AA•+ dimer is far higher, ca. 7. Theoretical calculations
also predict that base stacking of the cation radical over the
neutral adenine results in a high binding energy (12 to 16 kcal/
mol depending on level of calculation; see Table 2). Thus, the
stacked dimer cation radical system is predicted to be quite
stable, and this stabilization comes through the delocalization
of the charge over the two adenine bases. We calculate a
negligible barrier to deprotonation in the monomer cation radical
and a large barrier to deprotonation in the stacked systems (Table
3). These results are consistent with the calculated pKa’s of ca.
-0.3 and ca. 7. It is interesting to compare these values with
that of the parent adenine (pKa ca. 144). We see the pKa drops
by ca. 14 units for a full charge. The drop of ca. 7 pKa units
when the cation charge is shared over two adenines is therefore
expected.
A•+ (monomer)
e1c
-0.3d no barrier
-
stacked (AB-AB)s•+
8e
7
uphill process
12 to 16f
a B3LYP/6-31G* calculated with PCM solvent model. b See Figures
6 and 8. c See refs 3, 4. d Calculated for adenine cation radical.
e Suggested from present study. f At various levels of calculations (for
details see Table 2).
we found that A•+ deprotonates in the presence of water without
a barrier; however, in the stacked conformation a substantial
uphill process is involved in stretching the N6-H′ bond up to
1.45 Å. We conclude that the pKa of the A•+ monomer is quite
low while in the stacked system A•+ has a far higher pKa value.
When water molecules are considered explicitly, we find partial
localization of the spin; however, the charge resonance interac-
tions in the stacked system still significantly inhibit the
deprotonation reaction. The present study shows that the
deprotonation reaction from the A•+ monomer is facile in the
presence of a proton acceptor (such as, the first hydration shell),
while in the stacked system this deprotonation has a significant
uphill barrier.
4. Conclusions
(iii) Nature of charge delocalization in adenine stacks: We
note that the predicted nature of charge delocalization in stacked
adenine (see Figure 6b) is quite different that for guanine stacks
in which the charge is predicted to localize mainly on one of
The salient findings drawn from the present study are as
follows:
(i) Identification of A•+ and A(-H)• in model systems: In
this study, employing ESR spectroscopy and theoretical calcula-
9
J. AM. CHEM. SOC. VOL. 130, NO. 31, 2008 10291

A R T I C L E S
Adhikary et al.
the guanine bases.39a,40 Calculations predict a very small nuclear
relaxation energy (NRE) of ca. 0.10 eV on formation of the
adenine cation radical in the AT base pair.41 As a consequence,
the adenine cation radical has nearly the same structure as it
has in the neutral state. However, the corresponding NRE on
guanine cation radical formation in the GC base pair is far larger
(0.34 eV).41,42 Thus, adenine stacks in dsDNA are naturally
situated to delocalize charge (hole), whereas guanine stacks
undergo reorganization and localize the hole. Our calculations
show that the effect of the first hydration layer of solvent (18
waters) on the hole delocalization in the adenine dimmer cation
radical in a stacked geometry allows for polarization and partial
localization of the hole to favor one adenine (see Figure 7b);
yet even so, the stacked base moieties of the adenine dimer
cation radical would still share charge and remain temporarily
stable to deprotonation. Of course, in solution, some hindrance
to this delocalization of charge (hole) would be expected from
the solvent-induced polarization and the resultant partial local-
ization. The implications of these results suggest that, in DNA,
stacking will stabilize the adenine cation toward deprotonation
and that solvent dynamics and associated polarization of charge
will have a strong effect on the movement of the hole through
adenine stacks as has been suggested previously.27,42,43
Acknowledgment. This work was supported by the NIH NCI
under Grant No. R01CA045424. A.K. and M.D.S. are thankful to
the Arctic Region Supercomputing Center (ARSC) for a generous
grant of CPU time and facilities. A.K. is personally thankful to the
ARSC staff for their constant cooperation and timely help.
Computational studies were also supported by a computational
facilities grant NSF CHE-0722689.
Supporting Information Available: Figure S1 showing step-
wise formation of A•+ in a glassy sample. Figure S2 the ESR
spectrum for A•+ 8-D-dAdo with the simulated spectra of A•+.
Figure S3 gives the spectra and analyses of one-electron oxidized
dAdo at pH’s 7.5 and 8. Figure S4 shows formation of A(-H)•
on annealing A•+. Figure S5 compares results in 7.5 M LiCl
glasses with those found in 15 M LiCl glasses. Figure S6 shows
the sensitivity of the g value and A•+ stabilization with dAdo
concentration. Figures S7 and S8 show the B3LYP/6-31G*
optimized geometries of the various species considered in this
work. Figure S9 shows the calculated pKa values in an adenine
base cation radical in syn and anti conformations. Figure S10
shows the HF-MP2/6-31G*, BHandHYP/6-31G*, and HF/6-
31G* calculated spin density distributions in the base stacked
adenine dimer cation radical. Figures S11 and S12 show the
MP2/6-31G* optimized geometries of a stacked adenine dimer
cation radical and of a stacked neutral adenine dimer. Table T1
gives the hyperfine coupling constant (HFCC) values and spin
densities at various atoms of A•+ and of A(-H)• in different
conformations. Tables T2 and T3 present the details of pKa
calculations. Geom-1 to 12 represent the optimized geometries
of the various species considered in this study. Finally, a full
ref 30a is given. This information is available free of charge
via the Internet at http://pubs.acs.org/.
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I.; Nakamura, T.; Nakatani, K.; Yoshioka, Y.; Yamaguchi, K.;
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