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Decomposition of Thymidine by Low-Energy Electrons Implications for the Molecular Mechanisms of Single-Strand Breaks in DNA.

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DNA Strand Breaks
DOI: 10.1002/anie.200503930
Decomposition of Thymidine by Low-Energy
Electrons: Implications for the Molecular
Mechanisms of Single-Strand Breaks in DNA**
Sylwia Ptasińska, Stephan Denifl, Sascha Gohlke,
Paul Scheier, Eugen Illenberger,* and Tilmann D. M"rk
Low-energy electrons decompose gas-phase thymidine (a
thymine unit coupled to a sugar unit) through dissociative
electron attachment (DEA) involving two low-lying resonances at 1.2 and 1.8 eV and a broad resonant feature located
between 5.5 and 12 eV. The peak at 1.2 eV arises from the
rupture of the glycosidic N1C1 bond (observable through
the anion of the sugar moiety), whereas the resonance located
at 1.8 eV results from the loss of a neutral hydrogen atom
from the N3 position of the thymine moiety within thymidine.
The broad resonant feature in the energy range 5.5–12 eV is
associated with the rupture of the N1C1 bond, in this case
observable through the thymine anion generated. From the
analysis of the DEA signatures it follows that excess-electron
transfer from the thymine to the sugar unit within thymidine
and vice versa is not operative. These findings have significant
consequences for the molecular description of DNA damage
caused by low-energy electrons. It excludes the possibility of
electron migration from low-lying p* MOs of a nucleobase to
low-lying s* MOs of the DNA backbone leading to strand
The investigation of damage in biological systems, more
specifically, in living cells and DNA, induced by high-energy
quanta has been the subject of countless investigations over
the past decades.[1–3] Such alterations can appear, for example,
when human beings are exposed to radioactivity or other
sources of high-energy radiation. On the other hand, analogous questions arise in the application of radiosensitizers in
tumor therapy. It is well accepted that the main biological
effect is usually not produced by the primary interaction of
the high-energy quanta with the complex molecular network
in a living cell, but rather by the action of the secondary
species which are generated along the ionization track.[2] The
[*] Dipl.-Chem. S. Gohlke, Prof. Dr. E. Illenberger
Institut f(r Chemie und Biochemie
Physikalische und Theoretische Chemie
Freie Universit1t Berlin
Takustrasse 3, 14195 Berlin (Germany)
Fax: (+ 49) 30-838-56612
Mag. S. Ptasińska, Mag. Dr. S. Denifl, Prof. Mag. Dr. P. Scheier,
Prof. Dr. T. D. M1rk
Institut f(r Ionenphysik und Angewandte Physik
Leopold Franzens Universit1t Innsbruck und
Center for Molecular Biosciences Innsbruck (CMBI)
Technikerstrasse 25, A-6020 Innsbruck (Austria)
[**] Financial support from FWF (Wien), EU (Brussels) through the
EPIC Network and DFG (Bonn) is gratefully acknowledged.
Angew. Chem. Int. Ed. 2006, 45, 1893 –1896
interaction of these secondary species (ions, electron, or
radicals) with DNA and its surroundings can cause mutagenic, genotoxic, and other potentially lethal DNA lesions,[3]
such as single-strand breaks (SSBs) and double-strand breaks
(DSBs). Electrons are the most abundant of these secondary
species having an initial energy distribution extending to
about 20 eV.[4] For the understanding of the effects of
radiation in cells, it is therefore essential to investigate the
action induced by these electrons on vital cellular components
such as water and DNA.
So far the effects from high-energy radiation has been
investigated for two systems that vary greatly in complexity,
namely plasmid DNA and building blocks of DNA in the gasphase. Experiments on plasmid DNA demonstrated that
electrons at subionization energies induce SSBs and DSBs.[5]
Recent experiments showed the potential of electrons at even
subexcitation energy (0–4 eV) to induce SSBs in plasmid
DNA.[1] Although SSBs within double-stranded DNA is not
likely to be lethal for the cell, the situation may dramatically
change during the process of replication of the nucleic acid,
that is, when the double strand separates into two individual
On the other hand, gas-phase studies[6–12] revealed that
isolated nucleobases (NBs) undergo dissociative electron
attachment (DEA) in the energy range of approximately 6–
9 eV and also at subexcitation energies (< 3 eV, wherein only
SSBs are observed). The high-energy feature, however, leads
to loss of H and further fragment ions associated with the
rupture of the ring structure, whereas the low-energy
resonance exclusively leads to the loss of neutral hydrogen
through the DEA process shown in Equation (1).
e þ NB ! NB# ! ðNBHÞ þ H
NB# is the transitory negative ion (TNI) generated by
resonant electron capture and (NBH) is the closed-shell
anion formed by ejection of a neutral hydrogen radical from
NB#. The reaction is energetically driven by the appreciable
electron affinity of the (NBH) radicals that lie in the range
between 3 and 4 eV.[6, 10] Further experiments with partly
deuterated thymine[9, 11] demonstrated that hydrogen abstraction is operative exclusively from the N sites. Moreover, by
exactly tuning the electron energy, the loss of H can even be
made site selective: although electrons at 1 eV induce H loss
at the N1 position (N1H), the process can be switched, at
1.8 eV, to N3-H.[12]
These last findings were obtained by using thymine and
uracil, methylated at one of the N positions. The observation
that neither the loss of H from CH3, nor the loss of the entire
methyl group takes place when at the corresponding N
position H is replaced by CH3. These findings have significant
consequences for the molecular description of strand breaks
by low-energy electrons. A recent theoretical study[13] (modeling a section of DNA that contained cytosine, the sugar ring,
and the (neutralized) phosphate group) predicted a transfer
of the electron from the initial p* anion state of the base to a
s* state in the backbone, ultimately leading to rupture of the
CO bond between the phosphate and the sugar. Within
DNA the N1 position of T is coupled to the sugar moiety, and
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
according to the results obtained in the methylated compounds, transfer of energy via the N1 position is inhibited
once N1H is replaced by N1CH3.
Although the inhibition of energy transfer should also
apply if CH3 is replaced by the sugar, one cannot necessarily
exclude the transfer of excess charge, initially localized on
thymine, through the N1C1 glycosidic bond to the sugar
molecule, particularly when low-lying s* MOs are accessible.
To track this problem, we have studied herein low-energy
electron impact to gas-phase thymidine (Td), which represents a thymine coupled to the sugar 2-deoxyribose (DRB)
through a condensation reaction. For
simplicity, the deoxyribose moiety in
Td (DRBOH) will be assigned as dR
throughout this manuscript.
The present investigations were
performed in a crossed electron/molecule beams device at the Innsbruck
laboratory with a method previously
described in detail.[6, 14] The electron
beam is formed in a custom-built
hemispherical electron monochromator, operated at an energy resolution
between 110 and 130 meV (full width at half maximum) and
an electron current of 5–8 nA. The molecular beam emanates
from a source that consists of a temperature-regulated oven
and a capillary. Experiments have been performed in the
range of 390–426 K to obtain information on the thermaldecomposition behavior of Td. This was an inherent problem
in the herein described experiments and as such is discussed
below. Negative ions formed in the collision zone of the
crossed beams are extracted by a weak electric field towards
the entrance of the quadrupole mass spectrometer. The massselected negative ions are detected by a channeltron by using
a single-pulse counting technique. The intensity of a particular
mass-selected negative ion is then recorded as a function of
the electron energy.
The electron-energy scale was calibrated by using the
known SF6 signal near 0 eV. Absolute calibration of the
DEA cross sections were established by using the established
cross section of Cl/CCl4,[15] which yielded measures for the
cross sections at an estimated accuracy within one order of
magnitude. Thymidine was purchased from Sigma–Aldrich at
a stated purity of 99.5 %.
Figure 1 shows the energy dependence of negative ions
observed from electron impact to the gaseous sample
generated at 398 K. The signal at 241 amu can be assigned
to (TdH) owing to the loss of a neutral hydrogen atom
from thymidine according to Equation (2). This reaction is
analogous to that observed in the nucleobases [Eq. (1)] and
must proceed on an intact thymidine molecule.
Td þ e ! Td# ! ðTdHÞ þ H
sugar unit. The sugar unit is then further subjected to the loss
of two hydrogen atoms. These results are in agreement with a
previous study performed at the Berlin Laboratory[16] in
which questions were raised with respect to a possible thermal
decomposition of Td. A detailed study of that problem
revealed that the vast majority of the beam in fact consists of
nondecomposed Td molecules. As shown in Figure 1, the
dashed portions in the ion yields arise from electron attachment to products of thermally decomposed Td.
Irrespective of the presence of thermal decomposition,
the (TdH) signal (241 amu) must arise from intact Td
molecules. The site from which the hydrogen atom is
abstracted can immediately be identified through comparison
with the signal arising from hydrogen loss from thymine
methylated at the N1 position (m1T; Figure 2). In m1T,
hydrogen loss from the N1 position is blocked,[12] and the ion
signal (MH) (M corresponds to the target molecule) is
The signal at 125 amu can be assigned to (TH) and that
at 115 amu to (dR2 H) . Under the assumption that the
entire signal arises from reactions of intact Td targets, the
signals can then be attributed to the scission of the N1C1
bond with the excess electrons localized on the thymine and
Figure 1. Yields of product ions observed from electron attachment to
thymidine (Td, 242 amu) obtained at a temperature of 398 K. T
represents thymine and dR the 2-deoxyribose molecule after loss of
OH. The numbers on the intensity scale (I) correspond to the
estimated absolute DEA cross sections in units of 1022 m2. The
dotted portions of the ion-yield curves are identified to arise from
thermally decomposed Td (see the text).
Figure 2. Comparison of the yield of anion (MH) resulting from
hydrogen loss in thymidine (M = Td) with that of thymine methylated
at the N1 position (M = m1T).
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 1893 –1896
consequently owing to hydrogen loss from the N3 position. As
the shapes of the two signals in Figure 2 nearly coincide, we
can conclude that Td hydrogen abstraction predominantly
occurs at the thymine moiety, more precisely, at the N3
position. The small contributions near 1 and 0 eV may be due
to H loss from the sugar moiety.
Figure 3 shows the 125-amu signal and the 115-amu signal
on an extended energy scale. Although the shape of the lowenergy 125-amu signal (1) is very similar to that from isolated
Figure 4. The yield of (TH) from Td at an elevated temperature of
426 K.
Figure 3. Comparison of negative-ion formation from Td and the
isolated building blocks T[6] and 2-deoxyribose (DRB) [17] on an
extended energy scale.
T, it differs completely at higher energies (2) in that an
additional pronounced contribution in the range 5.5 to 10 eV
is observed only from Td. Superficially, from this additional
(TH) signal, one could conclude that the beam consists
only of nondecomposed Td molecules. The additional signal
would then also be evidence of the different probabilities to
distribute excess energy, that is, in isolated T, the excess
energy must be shared between H and (TH) , as the
negative ion is unstable with respect to dissociation and
autodetachment above 3–4 eV. This therefore makes DEA
unlikely. In contrast, in Td the excess energy can be shared
between polyatomic fragments of approximately equal size
with a correspondingly large number of degrees of freedom
resulting in the formation of (TH) at energies above 5 eV.
A careful analysis of the 125-amu signal at different
sublimation temperatures, however, revealed that the intensity ratio between features 1 and 2 changes with increasing
temperature in favor of the low-energy contribution 1, see
Figure 4. This directly indicates that the low-energy (TH)
signal arises from thermally decomposed Td (in part or
completely). Moreover, the shape of the 115-amu fragment
also shows a variation with temperature (not shown here) in
that the peak near 0 eV is relatively enhanced until, at 425 K,
the shape of the ion yield becomes very close to that from
isolated DRB. This also indicates thermal decomposition
leading to the 115-amu signal located close to 0 eV (in part or
Angew. Chem. Int. Ed. 2006, 45, 1893 –1896
The likely thermal-decomposition products are (TH)
and dR (rupture of the glycosidic N1C1 bond). Electron
attachment to neutral (TH), however, would never lead to a
(TH) signal like that shown in Figures 1 and 3.[18] A likely
scenario is that the thymine radical (TH), formed by
thermal decomposition, is transformed into T by hydrogen
pickup in the course of collisions with the walls of the
capillary. Accordingly, the complement dR (117 amu,
C5H9O3) is transformed into a closed-shell compound of the
stoichiometric composition C5H10O3 and subjected to DEA.
The alternate (and probably more likely) scenario is that
thymidine decomposes into T + C5H8O3 (116 amu); that is,
the cleavage of the N1C1 glycosidic bond is accompanied by
hydrogen transfer generating two closed-shell fragments
which are subjected to DEA.
Irrespective of the thermal-decomposition products, our
data indicate that near 400 K (the temperature at which the
spectra of Figure 1 and Figure 3 have been recorded), the vast
majority of the molecular beam consists of intact Td
molecules. This can be concluded directly from the intensity
ratio between feature 1 and feature 2 in the (TdH) signal in
Figure 3. As the cross section for DEA has, normally, a
general reciprocal dependence with energy,[18] the density of
the target creating feature 2 (Td) must be appreciably larger
than that creating feature 1. This also follows from the
absolute intensity of the (TH) signal, which, from T is more
than two orders of magnitude larger than from Td (at a
comparable gas pressure). It is in fact likely that the entire
low-energy feature of the (TH) signal originates from T
(present as a thermal-decomposition product) and not from
intact Td. This directly follows from the shape of the ion yield,
(MH) , owing to the loss of hydrogen from T that is
methylated at the N1 position (m1T) (see Figure 2). Methylation at N1 or coupling to the sugar should have the same
effect on T with respect to hydrogen loss. We therefore
conclude that the low-energy (TH) signal arises from
thermal decomposition. Similar arguments apply for the 115amu signal near 1.2 eV. This leads to the conclusion that the
structure near 0 eV is a result of decomposition, whereas the
majority of the signal intensity at 1.2 eV can be attributed to
the DEA reaction of intact Td. In Figure 1 the dotted part of
the ion-yield curve corresponds to contribution from thermaldecomposition products.
To summarize the present findings:
a) At energies above 5.5 eV, electron attachment to thymidine occurs through localization of the excess charge on
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
the thymine part. This is supported by the observation of a
similar feature in isolated T (yielding a variety of DEA
fragments and characterized as a core excited resonance[6]). The excited transient anion decomposes into
(TH) through rupture of the glycosidic N1C1 bond.
The neutral counterpart is C5H9O3 (dR), which may
further decompose (by loss of H2, H2O, etc.) due to the
presence of appreciable excess energy.
b) At subexcitation energies (< 3 eV), Td captures electrons
through resonances located at 1.8 eV and at 1.2 eV. The
1.8-eV feature ((TdH) at 241 amu) can be assigned as a
shape resonance with p* character, with the excess charge
localized on the thymine moiety leading the loss of H from
the N3 position. The 1.2-eV resonance ((dR2 H) at
115 amu) is linked to electron localization on the sugar
moiety (shape resonance of s* character) leading to the
rupture of the glycosidic N1C1 bond.
We have hence a situation wherein initial capture into s*
MOs at the sugar unit leads to rupture of the s (N-C) bond,
whereas capture into p* MOs of the thymine unit exclusively
leads to loss of H from the N3 site. This last reaction is
expected to occur through vibronic coupling, that is, a mixing
of p* states with repulsive valence s* states through vibrational motion.[19] Only core excited states of the thymine unit
induce cleavage of the glycosidic NC bond.
We shall now briefly consider the implications of these
findings for the molecular mechanism of DNA damage by
low-energy electrons. The fact that the 115-amu signal does
not carry any signature of electron attachment to the thymine
moiety and, accordingly, neither the (TdH) signal (loss of
hydrogen from N3) nor the (TH) signal carries any
signature of electron attachment to the sugar moiety. It
therefore follows that electron transfer between the two units
(associated with fragmentation) is inhibited. Rupture of the
N1C1 bond in fact takes place, but only through electron
localization on either of the two units.
The present findings directly demonstrate that migration
of the excess charge from the p* anions of the nucleobases to
the DNA backbone is inhibited and may hence not contribute
to SSBs as previously proposed.[13] Instead, attachment of
low-energy electrons can lead to loss of H from the N3
position (1.8 eV) and to the rupture of the N1C1 bond at
either 1.2 eV or in the energy range between 5.5 eV and 10 eV.
All three processes can be considered as primary events
contributing to strand breaks. The first creates a highly mobile
and reactive H radical (with its potential to induce further
damage in DNA), whereas the latter two induce rupture of
the glycosidic bond that can lead to excision of thymine from
DNA. It has to be noted that the presence of the phosphate
group could in fact modify the charge-transfer behavior with
respect to that presently observed in Td.
So far there is little information on the role of the
phosphate group. In a very recent study[20] on self-assembled
monolayers of DNA with orientations perpendicular and
parallel to the surface, the desorption of OH following
electron bombardment was studied. From the analysis of the
data the authors concluded that in the energy range 2–5 eV
direct DEA reactions to the phospate unit take place, whereas
at higher energy reactive scattering may also contribute to the
OH desorption signal.
Received: November 6, 2005
Keywords: anions · DNA damage · electron attachment ·
gas-phase reactions · nucleotides
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2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 1893 –1896
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implications, decompositions, thymidine, strany, molecular, electrons, break, low, mechanism, single, dna, energy
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