J. Kopyraa,
A. Kellerb and
I. Bald*cd
aDepartment of Chemistry, Siedlce University, 3 Maja 54, 08-110 Siedlce, Poland
bInstitute of Ion Beam Physics and Materials Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany
cInstitute of Chemistry – Physical Chemistry, University of Potsdam, Germany. E-mail: ilko.bald@uni-potsdam.de
dBAM Federal Institute of Materials Research and Testing, Berlin, Germany
First published on 6th January 2014
Gemcitabine (2′,2′-difluorocytidine) is a well-known radiosensitizer routinely applied in concomitant chemoradiotherapy. During irradiation of biological media with high-energy radiation secondary low-energy (<10 eV) electrons are produced that can directly induce chemical bond breakage in DNA by dissociative electron attachment (DEA). Here, we investigate and compare DEA to the three molecules 2′-deoxycytidine, 2′-deoxy-5-fluorocytidine, and gemcitabine. Fluorination at specific molecular sites, i.e., nucleobase or sugar moiety, is found to control electron attachment and subsequent dissociation pathways. The presence of two fluorine atoms at the sugar ring results in more efficient electron attachment to the sugar moiety and subsequent bond cleavage. For the formation of the dehydrogenated nucleobase anion, we obtain an enhancement factor of 2.8 upon fluorination of the sugar, whereas the enhancement factor is 5.5 when the nucleobase is fluorinated. The observed fragmentation reactions suggest enhanced DNA strand breakage induced by secondary electrons when gemcitabine is incorporated into DNA.
The LEE induced DNA strand breakage is directly enhanced by cisplatin and its derivatives.14 Furthermore, gas-phase DEA measurements have shown that cisplatin reacts with LEEs close to zero eV resulting in a release of both chlorine atoms after electron attachment thereby presumably facilitating the DNA binding.15 After activation, cisplatin binds to the N7 sites of neighboring guanine (G) bases resulting in intra- and interstrand cross links and subsequent cell damage.16
Halogenated uracils are incorporated into the DNA and lead to radiosensitization due to an enhancement of the electron attachment and dissociation cross sections.17 5-Bromouracil and 5-iodouracil appear to be particularly effective, since Br− and I− are formed by electron attachment at zero eV with cross sections of 4 × 10−14 cm2 and 9 × 10−14 cm2, respectively.7,18,19
Gem is a widely applied therapeutic due to its effectiveness towards a broad range of tumors. Although its biological effect as an inhibitor of DNA synthesis and repair is well-studied,2 a radiosensitization due to direct interaction of Gem with secondary electrons has never been considered. Gem is a derivative of 2′-deoxycytidine (dCyt), in which the two hydrogen atoms at C2′ are replaced by two F atoms (Fig. 1). The presence of F atoms usually increases the electron attachment cross sections of the respective molecule considerably. Therefore, in Gem we expect a specific radiosensitization of the sugar unit, as opposed to halogenated purines and pyrimidines, in which the nucleobase is sensitized.17
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Fig. 1 Molecular structures of 2′-deoxycytidine (dCyt), 2′-deoxy-5-fluorocytidine (dFCyt) and 2′,2′-difluorocytidine, which is referred to as gemcitabine (Gem). |
In a number of experimental and theoretical DEA studies on small DNA model compounds the question was discussed, whether strand breakage is due to initial electron attachment to the DNA nucleobases,20 or directly to the DNA backbone.21 For strand breakage to happen after LEE attack at the nucleobase the excess charge must be transferred to the sugar-phosphate backbone.22 Despite the large research effort, it could not yet be clarified satisfyingly, which pathway dominates. The most conclusive answer so far is that both mechanisms are operative at different energies as was demonstrated in a recent DEA study on the nucleotide 2′-deoxycytidine-5-monophosphate.23
Here, we probe the competing pathways of electron attachment to either the nucleobase subunit or the sugar unit by studying DEA to the nucleoside dCyt and compare the DEA spectra with the ones obtained from 2′-deoxy-5-fluorocytidine (dFCyt) and Gem (Fig. 1). Our comparative study demonstrates how the reaction pathways can be shifted by fluorination, thus opening up the possibility to control the reaction dynamics by rational chemical design. Furthermore, we demonstrate for the first time an enhanced reactivity of Gem towards LEEs which might contribute to its radiosensitizing properties. The improved understanding of the physicochemical mechanisms of radiosensitization will support a rational and thus efficient optimization of therapeutically used radiosensitizers.
From dCyt a fragment ion is observed at m/z 110, which corresponds to the closed shell dehydrogenated cytosine (C, C4H5N3O) anion [C–H]− (Fig. 2a). The ion yield curve in Fig. 2b shows that it is formed through a narrow resonance close to 0 eV, and with lower intensity at 1.5 eV. Similar signals close to 0 eV have been previously observed in electron attachment to the sugars 2-deoxy-D-ribose,25 D-ribose26 and D-fructose,27 the nucleotide 2′-deoxycytidine-5-monophosphate,23 and the DNA backbone surrogate D-ribose-5-phosphate.21 The near 0 eV signals are generally assigned to electron attachment to the sugar unit by initial formation of a dipole-bound doorway state.28 In this case the electron is initially trapped by the high dipole moment of the molecule, and the diffuse dipole bound state may result in dissociation if it couples to a valence bound state. In contrast, the resonance around 1.5 eV is most likely located at the cytosine unit and corresponds to the π* shape resonance observed from isolated cytosine at the same energy.29 Previous measurements on nucleoside analogues have shown that also in more complex systems the resonances of the isolated building blocks prevail.30 Another weak signal from m/z 110 is observed at 9.5 eV (see inset of Fig. 2), which is most likely due to a core excited resonance located at the cytosine subunit.
A corresponding anion was also observed from dFCyt and Gem. In the case of dFCyt the fragment anion is observed at m/z 128 since it additionally carries a fluorine atom. The dehydrogenated nucleobase anion was observed from all three compounds at the same electron energies, however, the intensity ratios of the two resonances are remarkably different (Fig. 2b). In dFCyt the intensity of the 1.5 eV resonance is considerably higher than in dCyt, and the count rates suggest that it is indeed an increase of the fragmentation rate from the 1.5 eV resonance rather than a simple change of the intensity ratio. The [C–H]− anion formed from Gem is formed with high intensity close to zero eV whereas the 1.5 eV resonance appears to be unchanged compared to dCyt. Comparing the three different compounds the energetic positions of the π* and the zero eV resonance does not change upon fluorination of the corresponding subunit, but the DEA cross section increases considerably. A direct comparison of the signal intensities of different compounds is difficult and might result in large errors. Nevertheless, the enhancement of DEA cross section for the formation of the dehydrogenated nucleobase anion upon fluorination can be quantified under the assumption that only the resonance associated with the fluorinated subunit is modified, whereas the DEA cross section for the other resonance remains constant. Under this assumption we obtain an enhancement factor of EF0 eV,Gem = 2.8 for the near 0 eV resonance in Gem, and EF1.5 eV,dFCyt = 5.5 for the 1.5 eV resonance in dFCyt.
In the case of the close to 0 eV resonance in Fig. 2 it should be noted that there is an efficient transfer of the excess charge from the sugar unit to the cytosine unit. That is, the fluorination at the sugar moiety in Gem leads to 2.8 times more effective electron attachment to the sugar while the charge gets localized on the cytosine moiety after dissociation of the N-glycosidic bond. Based on previous DEA investigations of phosphate containing compounds it is likely that in the presence of a phosphate group an electron transfer from the sugar unit to the phosphate group also occurs resulting in phosphoester cleavage representing a direct DNA strand break.21,23 Such a reaction is driven by the high electron affinity of the phosphate group.
The complementary anion of the dehydrogenated base is the deoxyribose anion (dR−, here it must be noted that in the deoxyribose anion formed from free deoxyribose there is one more hydroxyl group present, which is replaced by the nucleobase in nucleosides), which is not stable enough to be observable in the mass spectrometer.25 Nevertheless, as shown in Fig. 3, from Gem we observed a corresponding anion including the two fluorine atoms indicating that the fragment anion is stabilized by fluorine:
Gem + e− (0.5 eV) → C5H7F2O3−(m/z 153) + [C–H] |
This is plausible since fluorine generally increases the electron affinity of the fragments. The fluorinated sugar anion is observed with very low intensity at 0.5 eV. From dCyt and dFCyt a signal close to zero eV was detected that is due to additional loss of water and a hydrogen atom (Fig. 3):
dCyt + e− (≈0 eV) → C5H6O2−(m/z 98) + C + H2O |
dFCyt + e− (≈0 eV) → C5H6O2−(m/z 98) + FC + H2O |
In the case of dFCyt the signal is almost three times as intense as that of dCyt indicating an increase of the electron attachment cross section upon fluorination at the cytosine. A corresponding fragment was not observed from Gem, but instead a fragment anion at m/z 94 was observed with very high intensity, which is due to additional loss of C2H2O2 from the sugar moiety:
Gem + e− (≈0; 0.6 eV) → C3H4F2O−(m/z 94) + C + C2H2O2 |
C3H4F2O− is formed within a broad signal peaking close to 0 eV with a clear shoulder at 0.6 eV indicating that the signal consists of contributions from different closely spaced resonances. Alternatively, the fragment ion at m/z 94 can also be assigned to the sum formula C4H3N2O− that can be formed by cleavage of the N-glycosidic bond and excision of the NH2 group and with the excess electron residing on the cytosine subunit. A similar fragment was previously observed from isolated cytosine, but at higher energies (5.2 eV and 7.3 eV).31
At m/z 115 another fragment ion is formed from all three investigated molecules that can be ascribed to N-glycosidic bond cleavage, formation of F2/H2 from the sugar ring and charge retention on the remaining sugar moiety. The corresponding ion yield curves are displayed in Fig. 4b, and all three curves show a narrow peak around zero eV and an additional signal from 0.5–1.5 eV (peaking at 1 eV from dCyt, at 1.1 eV from dFCyt and at 0.9 eV from Gem). The same fragment anion was observed in electron attachment to 2′-deoxyribose with similar resonant features close to 0 eV and 1.5 eV.25 This confirms that this fragmentation reaction is confined to the sugar moiety. It is important to emphasize that in this case also the 1 eV resonance is attributed to electron attachment to the sugar moiety (thus corresponding to a σ* resonance). Also this fragment anion is observed with higher intensity from Gem.
In all three molecules a series of small fragment anions (CN−, OH−, O−/NH2− and H−) is formed from different resonances at a variety of energies. While the first two fragment anions from all investigated molecules are generated in the low energy domain, more specifically at around 2 eV (CN−) and 0.3 eV (OH−), the H− anion is generated in the high energy range between 4.5 and 8 eV (not shown). In Fig. 5 the ion yield curves of the fragment anion at m/z 16 is displayed, which can be attributed to the isobaric anions O−/NH2−. They arise from a series of higher-lying core-excited resonances. These TNIs are formed when the incoming electron induces electronic transition and is concomitantly trapped in the field of electronically excited molecule. The first resonance between 6 and 8 eV matches the energy of the first resonance of O− reported from 2′-deoxyribose.25 On the other hand, the second resonance visible between 8 and 11 eV reflects the energy of resonances observed in both components namely 2′-deoxyribose and cytosine.25,29 Thus it is very likely that both the O− and the NH2− anion contribute to the intensity of the second resonance and arise from both sub-units.
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