DNA Backbone Radicals: ESR Characterization

1
Electrons and DNA From Electron Attachment to
DNA Strand Breaks
Michael Sevilla Chemistry Department Oakland
University
2
DNA electron and hole scavenging

• G, CH, T, Sug

• CH, T, hole scavenger

• G, S, electron scavenger

• electron and hole scavenger S

3

What are the mechanisms of electrons reaction
with DNA?

e-
e-

4
DNA and Electrons

• A variety of studies show that aqueous electrons
  add to the DNA bases and do not cause strand
  breaks.
• The base anion radicals undergo protonation
  reactions.
• Only nonsolvated electrons with kinetic energy
  (LEE) cause DNA strand breaks.
• At low temperatures electron in DNA are stable
  for long times.

5
Electron Tunneling b Decay Constant in DNA at 77
K
b (Å-1) 0.75 0.92 1.4 2.8

pdAdTpdAdT
DNA (salmon sperm)
pdIdCpdIdC
e-
Vacuum
k 1011exp(-bD)
Z Cai and M Sevilla, in "Topics in Current
Chemistry 237 Long Range Transfer in DNA II",
Gary Shuster, Ed., Springer-Verlag, pages
103-128, 2004 .
6
DNA g-irradiation Studies

• g-Irradiate hydrated DNA at 77K in frozen
  solutions
• Direct Ionization of DNA results
• Identify initial radicals by Electron Spin
  Resonance Spectroscopy

Ionizing radiation
DNA
DNA DNA
Oxidative path
Reductive path
"The Chemical Consequences of Radiation Damage to
DNA" D. Becker and M. D. Sevilla, in Advances in
Radiation Biology, Vol.17, (J. Lett, Ed.)
Academic Press, 121-180 (1993).
7
RADICAL COMPOSITION (g-irradiated DNA) ESR
Spectra

• The composition of DNA radicals (77 K) is ca.
• 25 (C-) C(N3)H
• 25 T-
• 40 G
• 10 Sugar
• All to 5

C(N3)H
8
Effect of scavengers on G-values
Sug
L. I. Shukla, R. Pazdro, D. Becker and M. D.
Sevilla, Radiation Research Vol. 163, 59-602
(2005).
9
Hole induced Sugar radicals in DNAOxidative
Pathway
C5
C1

C3
C4 ?
10
Oxidation pathway formation of neutral sugar
radical(s) from sugar cation radical(s)

• Deprotonation from C1, (C2), C3, C4, C5 may
  occur.

L. I. Shukla, R Pazdro, D Becker and M D
Sevilla, RAD. RES.163, 591602 (2005)
11
How do electrons damage DNA?Clues fromIon Beam
Irradiation of DNA

• ca. 100 MeV/nucleon
• O8, Ar18, Kr36, Xe54

D Becker, A Bryant-Friedrich, C Trzasko and M
D Sevilla, Rad Res 160, 174 (2003)
12
PARTRAC Calculations, Hauptner et al (2006)
13
Cross Section of Heavy Ion Track
Aloke Chatterjee and John Magee, J. Phys. Chem.
Vol. 84, 3629-3536 (1980)
14
High Yields of Sugar radicals found in ion-beam
irradiated DNA
Excited States Important?
D. Becker, A. Bryant-Friedrich, C. Trzasko, and
M. D. Sevilla, Radiation Research, 160, 174-185
(2003) M. Bowman, D.Becker, M. D. Sevilla and J.
Zimbrick, Radiation Research Vol. 163, 447-454
(2005).
15
Yields of Sugar Radicals Increased in core of
ion beam irradiated DNA

• ? Sugar Radical Yield per Joule in track core
  more than in the penumbra.
• ? New sugar phosphate species found in ion beam
  irradiated DNA

D. Becker, A. Bryant-Friedrich, C. Trzasko, and
M. D. Sevilla, Radiation Research, 160, 174-185
(2003)
16
Ar ion Irradiated DNAPhosphoryl radicals -
strand break radical(s)
17
2nd Strand Break radical found in ion beam
irradiated DNA
N from DNA
C3dephos?
Simulation using theoretical spectral parameters
18
LEE Induced Strand Scission
Path B bond scission
Path A bond scission
LEE
D. Becker, A. Bryant-Friedrich, C. Trzasko, and
M. D. Sevilla, Radiation Research, 160, 174-185
(2003)
19

• What about Excited states?
• We know excitation of DNA holes forms sugar
  radicals

20
Visible excitation converts G to Sugar Radicals
A Adhikary, S Collins, D Khanduri, and M D
Sevilla, JPC B 2007, 111, 7415-7421
A Adhikary, A Kumar
and M D Sevilla, Rad Res 2006 165, 479484
21
LEE Reaction with DNAStrand Break Formation

• LEE capture at the base or the sugar phosphate
  backbone?
• What energies are needed?
• Are excited states involved?

22
DNA Strand break induced by low energy electrons
F. Martin, P.D. Burrow, Z. Cai, P. Cloutier, D.J.
Hunting, L. Sanche, Phys. Rev. Lett. 93,
068101-1 (2004)
B. Boudaiffa, P. Cloutier, D. Hunting, M. A.
Huels, L. Sanche, Science 2000, 287, 1658
23
Modeling the LEE Induced DNA Strand Break
Sugar-Phosphate-Sugar model

• Li, X. Sevilla, M. D. Sanche, L. J. Am.
  Chem. Soc., 2003, 125, 13668

DNA
24
The Potential Energy Surfaces
DNA
25
Basis set dependence of Spin Distribution
3-21G(d) 6-31G(d)
6-31G(d)
Xifeng Li and Michael D. Sevilla, in
Theoretical Treatment of the Interaction of
Radiation with Biological Systems. J. R. Sabin
and E. Brandas, Eds., Advances in Quantum
Chemistry, Elsevier, Volume 52, 59-88 (2007).
26
New DFT Calculations on 5-dTMPH Anion Radical
Recent Previous Work J. Simons, Acc. Chem.
Res. 2006, 39, 772-779 Bao, X. Wang, J. Gu,
J. Leszczynski, J. Proc. Nat. Acad. Sci.U.S.A.
2006, 103, 5658. Gu, J. Wang, J. Leszczynski,
J. J. Am. Chem. Soc. 2006, 128, 9322.
Adiabatic Barriers of 7 kcal/mol for 3- C-O bond
and 14 kcal/mol for 5-C-O bond

• What about the vertical surface?
• A. Kumar M. Sevilla, J. Phys. Chem. B 2007, 111,
  5464-5474

27
LEE Induced strand break in 5'-dTMPH as a
model Vertical or Adiabatic
pathways?
vertical
adiabatic
28
B3LYP/6-31G calculated adiabatic and vertical
potential surfaces (PES) of C5'-O5' bond
dissociation of 5'-dTMPH radical anion. The
singly occupied molecular orbital (SOMO) is also
shown
kcal/mol
26.0
B3LYP/6-31G
25.5
24.0
24.4
22.0
Vertical PES
22.5
20.0
20.7
18.0
17.6
16.0
16.5
TS
15.5
14.0
14.8
12.0
1.78 Å
10.0
10.8
Adiabatic PES
8.0
6.0
4.0
4.8
2.0
0.0
0.7
-7.4
-8.0
1.45 1.5 1.6
1.7 1.8 1.9
2.0
C5'-O5' (Å)
29
B3LYP/6-31G calculated adiabatic and vertical
potential energy surfaces (PES) of C5'-O5' bond
dissociation of 5'-dTMPH radical anion.
kcal/mol
22.0
B3LYP/6-31G
21.7
20.0
19.1
18.0
8.2 kcal/mol
Vertical PES
18.0
16.0
14.0
TS
14.4
13.5
12.0
1.78 Å
10.0
10.9
10.2
8.0
Adiabatic PES
6.0
5.7
4.0
4.8
4.5
2.0
0.0
0.5
0.6
-2.0
-4.0
-5.8
-6.0
1.45 1.5 1.6
1.7 1.8 1.9
2.0
C5'-O5'(Å)
30
B3LYP/6-31G optimized geometries of neutral and
anionic radical of 5'-dTMP with Na and 11
H2O. A. Kumar M. Sevilla, J. Phys. Chem. B
2007, 111, 5464-5474
Na
Na
5'-dTMPNa 11 H2O (Neutral)
5'-dTMPNa 11 H2O (Anion radical)
31
DFT (B3LYP/6-31G) calculated adiabatic
potential energy surface (PES) of C5'-O5' bond
dissociation of hydrated (11 H2O) 5'-dTMP radical
anion with Na as a counter ion.
Na
kcal/mol
33.0
B3LYP/6-31G
30.0
Na
27.0
28.9
24.0
Na
21.0
18.0
15.0
15.4
12.0
Na
Na
11.0
9.0
6.0
3.0
0.0
-3.3
-6.0
1.5 1.6 1.7 1.8
1.9 2.0 2.1 2.2 2.3 2.5
C5'-O5' (Å)
32
Proposed mechanism of single strand break (SSB)
due to attachment of LEE with 5'-dTMPH molecule
33
Why C-O not P-O?

• Experimentally only cleavage at the C-O bond is
  found.
• Y Zheng, P Cloutier, D Hunting, J R Wagner,
  L Sanche, JCP, 124, 064710 (2006).
• The loss of phosphate anion is the driving force
  for the reaction because the phosphate radical
  has a such a large EA.
• J. Simons, Acc. Chem. Res. 2006, 39,
  772-779

34
Base Release in Nucleosides Induced by Low
Energy Electrons
X. Li, L. Sanche and M. Sevilla, Radiation
Research, 165, 721 (2006) J. Gu, Y. Xie and H.
F. Schaefer, III, J. Am. Chem. Soc. 127,
10531057 (2005).
35
Potential Energy Surfaces (PESs) for C-N bond
dissociation in Nucleoside Anion Radicals
Calculated with DFT (b3lyp, 6-31G(d))
dT and dC Anion Radicals
dA and dG anion radicals
X. Li, L. Sanche and M. Sevilla, Radiation
Research, 165, 721 (2006)
36
LEE Induced Excited States

• LEE induced resonances found experimentally
  suggest excited state involvement.
• What are the available excited state levels?
• We performed TD-DFT calculations to aid our
  understanding in 5-dTMP-?

37
Excited States of Sugar Phosphate Portion
J. Simons, Acc. Chem. Res. 2006, 39, 772-779 J.
Berdys, I. Anusiewicz, P. Skurski, J. Simons JACS
(2004)
38
Molecular orbital Energies and MO plots of
neutral 5'-dTMPH. B3LYP/6-31G. Scaled values
by method of Modelli and Jones JPC 2006.
Experimental VOEs of thymine (Aflatooni et al.
J. Phys. Chem. A (1998) 102, 6205 )
MOs
Orbital Energy (eV) Scaled VOE (eV)
1.78 2.64
LUMO 4 (s3)
1.27 2.23
LUMO 3 (s2)
0.73 1.80
LUMO 2 (s1)
0.43 1.56 (1.71)
LUMO 1 (p2)
-0.84 0.53 (0.29)
LUMO (p1)
-6.24
HOMO (p)
ca. - 5 eV
39
Summary

• Strand break formation by LEE induced C-O bond
  scission is the lowest energy pathway in
  comparison to C-N, N-H or C-H bond scissions.
• LEE induced anion excited states likely provide a
  facile route to strand breaks.

40
DNA Radiation Chemistry Group at Oakland
University
Ph.D. Students.
Amitava Adhikary Deepti
Khanduri Anil Kumar Sean
Collins David Becker
Alyson Engle Lata Shukla Tom Casey
Collaborators
Leon Sanche Xifeng Li Acknowledgments This
research is supported by NIH NCI RO1CA045424
41
OU ESR GROUP