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Energy Transfer in Microhydrated Uracil, 5-Fluorouracil and 5-Bromouracil Jan Poštulka, Petr Slavicek, Juraj Fedor, Michal Farnik, and Jaroslav Kocisek J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07390 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 2, 2017

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The Journal of Physical Chemistry

Energy Transfer in Microhydrated Uracil, 5-Fluorouracil and 5-Bromouracil J. Poštulka,† P. Slavíček,∗,†,‡ J. Fedor,‡ M. Fárník,‡ and J. Kočišek∗,‡ †Department of Physical Chemistry, University of Chemistry and Technology, Technická 5, Prague 6, Czech Republic ‡J. Heyrovský Institute of Physical Chemistry v.v.i., The Czech Academy of Sciences, Dolejškova 3, 18223 Prague, Czech Republic E-mail: [email protected]; [email protected]

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Abstract Experiment and theory are combined to study the interaction of low energy electrons with micro hydrated uracil and its halogenated analogues 5-fluorouracil and 5bromouracil. We report electron ionization (EI) and electron attachment (EA) mass spectra for the uracils with different degrees of hydration. Both, EI and EA lead to evaporation of water molecules. The number of evaporated molecules serves as a measure of the energy transferred to the solvent. Upon EI, the amount of the energy transferred to neighboring water molecules is similar for all three studied species. On the other hand, the energy transferred upon EA rises significantly from uracil to 5-fluorouracil and 5-bromouracil. 5-bromouracil is the only studied molecule that undergoes dissociative electron attachment after hydration at the studied energy of 1.2 eV. Theoretical modeling of the energetics for the electron attachment process allows for setting the energy transferred to the solvent on the absolute scale. We discuss the importance of this energy for the radiosensitization.

Introduction Uracil (U), the smallest nucleic acid base, does not occur naturally in DNA; yet, it can be formed in DNA by de-amination from cytosine. Such a process leads to mutagenesis, 1 which is in higher organisms efficiently repaired (e.g. ref. [ 2]). Halogenated uracils 5-fluorouracil (FU) and 5-bromouracil (BrU) serve as artificial mutagens. FU is now routinely used as a cancer chemotherapeutics. 3 The modes of action for a brominated analogue of uracil seem to be similar. 4–6 In clinical practice, bromouracil is used in its nucleoside form. Halogenated uracils are now extensively studied in the context of concomitant chemoradiation therapy (CCRT) of cancer. The main advantage of the CCRT is the so called synergistic or supra-aditive effect: the combined effect of radiation and chemotherapy is bigger than the additive effect of the individual treatments. The synergistic effect of the FU is generally assigned to the antimetabolic action of the drug in the already irradiated 2


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tissue. FU inhibits the tumor cell division by its incorporation into DNA during the replication process 7–10 while its administration is more efficient in the cells damaged by ionizing radiation. Therefore, the interaction of the ionizing radiation with the FU seems to be insignificant for the observed chemoradiation synergy. In contrast to FU, the interaction of ionizing radiation with BrU and its metabolites is crucial for BrU synergistic action. The subject was first explored by pulsed radiolysis 11,12 proposing the halouracil anion stability in the aqueous solution as the main cause of the difference between the BrU and FU activity. These early studies sparked a continuous interest in the interaction of halogenated uracils with low energy electrons, which are believed to be the key species in the CCRT synergy (see e.g. [ 13–16]). The Uracil-5-yl. radical formed by Br dissociation from BrU via dissociative electron attachment (DEA) has been shown to produce single and double strand breaks in DNA. 17 Chomicz and co-workers 18 predicted radio sensitizing effectiveness for a range of uracil derivatives on the basis of the DEA mechanism. The necessary condition identified by Chomicz and coworkers is the effective formation of a free reactive Uracil-5-yl. upon the interaction with solvated electrons in a biological solution. This radical can cause damage to DNA directly via abstraction of hydrogen from the sugar moiety. An alternative explanation by Kumar and Sevilla 17 is based on the interaction of Uracil-5-yl. with water which forms a stable uracil molecule and a reactive OH radical that can cause the damage. Here, we demonstrate that in contrast to U and FU, microhydrated BrU dissociates upon the interaction with free low energy electrons. Therefore we experimentally confirm previous theoretical works. 18–20 However, in agreement with our work, 21 we also observe effective anion stabilization via energy transfer to the solvent. Therefore, in the present work, instead of focusing on the question "How does the solvent influence the interaction with low energy electrons?" we ask "How does the reaction of uracils with low energy electrons influence the solvent?". We combine mass spectrometry experiments and ab initio theoretical modeling to support our conclusions. We show that the amount of energy transferred into the environment is

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rather different for various substituted uracils at least for small cluster models.

Methods Experimental The experimental part was conducted on the cluster beam (CLUB) setup. 22 Configuration of the experiment was identical to the description given in Ref. [ 21]. The sample molecule was sublimated in the oven in front of the 90µm conical nozzle and expanded to the vacuum together with the He buffer gas. To induce microhydration of the molecules, ESI-Pergo gas humidifier was added into the He line consisting of a Nafion membrane tube placed in the temperature controlled reservoir with deionized water. This configuration enabled high control and stability of the hydration. The conditions used for different molecules are summarized in the Table 1. Before the experiments, samples were kept at 420 K under He atmosphere for 1 to 3 hours to evaporate the water and other volatile impurities. Table 1: Temperatures of the sample reservoir Tr and expansion nozzle Tn used for different molecules. Samples were purchased from Sigma-Aldrich, with stated purities SP. The temperature of the water reservoir was kept at 302K, stagnation pressures are shown with individual spectra. sample species

SP [%]

Tr [K]

uracil 5-fluorouracil 5-bromouracil

99 99 98

463 463 468 473 460 465

Tn [K]

The molecular beam was probed ∼1.3 m downstream the nozzle. Molecules collided with electrons at a controlled energy and product ions were analyzed by a bipolar reflectron TOF mass spectrometer. For the given expansion conditions (Table 1), we always measured mass spectra of cations at the incident electron energy of 70 eV and mass spectra of anions at the incident electron energy of 1.2 eV. This energy was chosen, because below this value the electron current in the present experiment can not be well controlled, as it was discussed in 4


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the previous study. 23 For the low energy resonances peaking bellow this value, 1.2 eV always represents the maximum of the anion signal.

Simulations We focus on the energetics of the electron attachment reaction

A + e− (1.2eV ) → A−

(1)

For uracil and halogenated uracils, we calculated adiabatic electron affinity (AEA) as a difference between the energy of the optimized structures of the anion and the neutral system. We then estimated the total energy transfer (TET) of reaction (1) as

T ET = Ee + AEA

(2)

assuming the kinetic energy of the incident electron Ee to be 1.2 eV. We considered either an isolated base (uracil, 5-fluorouracil or 5-bromouracil) or a cluster of a base with 3 or 5 water molecules. For all of the clusters, we evaluated the distribution of the T ET s, taking into account geometry variation due to the thermal nuclear motion of the particles via molecular dynamics (MD) simulations. The nuclear delocalization is an important aspect here as the width of the spectra are comparable to the energies of the processes under study. We constructed a distribution of the observable quantities via a reflection principle, 24,25 e.g. for the distribution of total energies, we numerically constructed an integral

σ(E) =

Z

~ ~ − E)dR ~ ρ(R)δ(T ET (R

(3)

~ at finite temperature T were evaluated within the MD where the nuclear densities ρ(R) simulations. In other words, the energy quantities were calculated along the MD trajectory. The MD simulations were performed for each cluster size for 25 ps, using a time step of 0.5 5



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fs. Each 25th frame was then selected for the recalculation. The simulation was performed at the constant temperature of 200 K, which was maintained by the Nosé-Hoover thermostat. The equations of motion were integrated with the RESPA algorithm. 26 The forces were calculated with the PBE functional with 6-31g* basis, augmented by the Grimme’s G3 correction to account for dispersion interaction. 27 The bromine atom was described with LANL2DZ effective core potential with a corresponding basis set. 28 The vertical electron affinities and the reorganization energies were recalculated at the BMK/6-31++g** level. 29 For a comparison, we also calculated the AEA together with the vertical electron affinity (VEA, calculated as a difference between the energy of the neutral system and the anion in the geometry of the neutral system) and the reorganization energy (λ, defined as the difference between the energy of the anion in the geometry of the neutral system and the energy of the anion in the equilibrium anionic geometry) for the minimum energy structures. The geometries of the optimal clusters are shown in Table 2 and the respective Cartesian coordinates are collected in the Supplementary material. We also calculated the binding energies of water molecules in these clusters and their dependence on the cluster size. These quantities allow us to compare the calculated energy released upon the electron attachment resulting in the water evaporation observed in the experiment. The structure and energetics for the minimum energy structures were calculated at the BMK/6-31++g** level, using the G3 dispersion correction. 27 The electronic structure calculations were performed in the Gaussian 09 code (revision D.01) 30 or with the GPU-based Terachem code. 31,32 The MD simulations were performed in the in-house Abin code. 33

Results and Discussion We organize the results as follows: we start with a discussion of the stability of the three molecules upon the electron attachment. We particularly focus on the question whether the

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Table 2: Minimum energy structures for small clusters of substituted uracils and water. For each cluster, we present also the vertical electron affinity V EA, the reorganization energy λ, the total energy for reaction (1) T ET and the water binding energy EB . All these energies are calculated at the BMK/6-31++g** level.

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fragmentation of the molecule is suppressed by the solvent. Next, we address the question how the energy released during the electron attachment dissipates into the environment. We then compare the energetics of the electron attachment and positive ionization. Finally, we discuss the possible implications of the present work for the chemoradiation cancer therapy.

Molecular fragmentation after electron attachment Low energy electrons efficiently attach to isolated U and FU and dissociate via loss of a hydrogen atom to form (M-H)− dehydrogenated parent anions. 34–37 Isolated BrU does not form such anions, but rather stabilize to the BrU− anion or dissociate via a release of the Br− or Uyl− anions. 38,39 As we observe in the present work, these processes are strongly affected by the solvent water molecules. The EA mass spectra for microhydrated uracils (U, FU and BrU) are shown on the right side of Figures 1, 2 and 3, respectively. For each molecule, we present 4 spectra at different expansion conditions corresponding to a different level of hydration, i.e. a different number of water molecules n in the neutral precursor cluster X(H2 O)n . The hydration level rises from the bottom to the top of the figure. Microhydration - adding a few water molecules to U or FU - results in a complete closing of the (M-H)− dissociation channel. We can not see this type of anions even at the lowest hydration conditions (Fig. 1,2 bottom right panel). The fragmentation is not fully closed for the BrU molecule. Even at the highest hydration conditions studied in this work (see Fig. 1 right panel - 2 bar), the BrU molecule dissociates via the Br− anion release. However, the relative intensity of the Br− signal decreases with the number of water molecules n in the BrU(H2 O)n cluster. We could not reach higher hydration conditions in the present configuration of the experiment to test if the dissociation channel closes completely. From the energetic point of view, it has been calculated by Chomicz and coworkers that the difference between stabilization and dissociation channels in bulk water is only 7.7 kcal/mol (∼0.3 eV), 18 our calculations at the BMK/6-31++g** level indicate the difference to be ∼0.1 eV. Therefore, there is no strong driving force for the bromide 8


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dissociation. Present experiments clearly show that the BrU anion stabilization becomes dominant with increased hydration of the clusters. The observation of Br− released from the cluster suggests the orientation of the bromine atom outside of the water clusters. This is consistent with the minimum energy structures identified in the present work. Table 2 shows calculated structures of the minimum energies of different small clusters of the three uracils. We observe that for all three uracils, water molecules first accumulate in the pockets around the nitrogen atoms. Such structures were previously proposed also on the basis of cavity ring down spectroscopy of uracil water complexes. 40 The nitrogen atoms represent the hydrogen dissociation sites after the electron attachment to U 41 and analogically to FU. The bromine atom points outside of the cluster even for 5 attached water molecules and the atom can therefore freely dissociate. Closing the BrU dissociation channel for larger clusters indicates that the stabilization is caused by kinematic caging of the outgoing fragment which in agreement with the hypothesis presented in our previous work on uracil and thymine. 21 Recent low energy electron scattering calculations of DNA bases in water 42,43 also support the conclusion that the caging is responsible for the observed closing of the dissociation channels. For both types of proposed electron attachment resonances at very low energies (σ ∗ 42 and π ∗ 43 resonances), the calculations show an increase in the attachment cross section. This should consequently increase the molecular dissociation. Such an effect is, however, not observed. Despite the fact that the cross section for the electron attachment of hydrated uracils is probably larger than for isolated ones, the dissociation is suppressed. We can conclude that the free electron attachment to microhydrated uracils primarily results in stabilization of the formed complex molecular anion. This process will be discussed in the following parts.

Energy transfer to solvent after electron attachment Expansion conditions in our experiment are set to form exclusively clusters containing only a single uracil molecule at a different level of hydration. At such conditions, the number of 9



Uracil

U+

U(H2O)

120

140

30 U(H2O)-n

2 bar

+ n+

HU(H2O)

200

0 100

negative MS ~1.2 eV

positive MS ~70 eV

400

+ m+

160

n+=0-5 m+=0-3 =0.67

180

U(H2O)

10

-

0 220 100 120 140 160 180 200 220 240

200

40 1.5 bar n+=0-4 m+=0-2 =0.43

200

0 100

120

140

160

180

1.5 bar n-=1-5 =2.4

20

0 220 100 120 140 160 180 200 220 240

200

320

12 1 bar n+=0-3 m+=0-2 =0.25

160

0 100

120

140

160

180

200

1 bar n-=1-3 =1.7

6

0 220 100 120 140 160 180 200 220 240 24

240

0.8 bar n-=0-2 =1.2

0.8 bar n+=0-2 m+=0-1 =0.1

120

0 100

2 bar -

n-=1-7 =3.1

20

400

Signal cpm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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120

140

160

180

200

12

U-

220 100 120 140 160 180 200 220 240

m/z

Figure 1: 70 eV electron impact ionization mass spectra of uracil (left) and 1.2 eV electron attachment spectra (right). The increase in the expansion pressure from bottom (0.8 bar) to the top of the figure (2 bar) results in the attachment of more water molecules on uracil in the expansion - higher level of microhydration. the evaporated water molecules can be used as a measure of the excess energy released to the solvent. The present experiment does not allow for the absolute measurement of the number of evaporated water molecules, however, we can estimate this quantity from the comparison of EI with EA. The mass spectra for the microhydrated U, FU and BrU corresponding to the different ionization mechanisms are shown in Figures 1, 2 and 3 respectively. The EI mass spectra are shown on the left figure panel while the EA spectra are shown on the right panel. The individual spectra are dominated by parent non-fragmented uracils with attached n+ or n− 10


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5-Fluorouracil

+

FU

FU(H2O)

240

HFU(H2O)

16 FU(H2O)n-

2 bar

+ n+ + m+

n+=0-4 m+=0-3 =0.55

120 0 120

negative MS ~1.2 eV

positive MS ~70 eV

360

8

140

160

180

0 220 120 140 160 180 200 220 240 260

200

40 n+=0-3 m+=0-2 =0.31

160

140

160

180

20

36 1 bar n+=0-2 m+=0-1 =0.17

240 120

140

160

180

18

1 bar n-=0-2 =0.58

0 220 120 140 160 180 200 220 240 260

200

120

20 0.8 bar n+=0-1 m+=0 =0.05

60

0 120

1.5 bar n-=0-4 =1.3

0 220 120 140 160 180 200 220 240 260

200

360

0 120

n-=0-6 =2.2

-

1.5 bar

0 120

2 bar -

FU

320

Signal cpm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


140

160

180

200

10

0.8 bar n-=0-1 =0.24

0 220 120 140 160 180 200 220 240 260

m/z

Figure 2: 70 eV electron impact ionization mass spectrum of 5-fluorouracil at different levels of hydration (left) and 1.2 eV electron attachment spectra at the corresponding expansion conditions (right). The increase in the expansion pressure from bottom (0.8 bar) to the top of the figure (2 bar) results in the attachment of more water molecules on 5-fluorouracil in the expansion - higher level of microhydration. − water in cations X(H2 O)+ n+ or anions X(H2 O)n− , respectively. For all three molecules, EI is

dominated by the molecular cation (n+ =0). This stems from a significant water evaporation from the precursor cluster, as confirmed by comparison with the EA spectrum. For example, the maximum number of water molecules n− for the U(H2 O)− n− anion is 7 (Fig.1), while the n+ is only 4 for the cationic U(H2 O)+ n+ clusters. The comparison between U, FU and BrU (Fig. 1,2 and 3, respectively) shows that water evaporation upon the EI is similar for all three molecules, while EA leads to a significantly different evaporation. 11



5-Bromouracil +

BrU

200 100

negative MS ~1.2 eV

positive MS ~70 eV

300

BrU(H2O)

+ HBrU(H2O)m

18

-

2 bar

+ n+ +

n+=0-7 m+=0-4 =0.72

12

BrU(H2O)n-

Br

320

2 bar

-

n-=0-4 =1.1

90

120 150 180 210 240 270

24 1.5 bar

160

BrU

6

0 0 180 200 220 240 260 280 300 320 60

Signal cpm

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1.5 bar 18

n+=0-4 m+=0-2 =0.37

n-=0-3 =0.44

12 6

0 0 180 200 220 240 260 280 300 320 60

90

120 150 180 210 240 270

240

120

1 bar n+=0-2 m+=0 =0.14

n-=0

=0

6

0 0 180 200 220 240 260 280 300 320 60

90

120 150 180 210 240 270

15

180 0.8 bar 90

1 bar

12

n+=0-2 m+=0 =0.09

0.8 bar

12

n-=0 =0

9 6

3 0 0 180 200 220 240 260 280 300 320 60

90

120 150 180 210 240 270

m/z

Figure 3: 70 eV electron impact ionization mass spectrum of 5-bromouracil at different levels of hydration (left) and 1.2 eV electron attachment spectra at the corresponding expansion conditions (right). The increase in the expansion pressure from bottom (0.8 bar) to the top of the figure (2 bar) results in the attachment of more water molecules on 5-bromouracil in the expansion - higher level of microhydration. What is the reason for the differences in the EA spectra? A simple explanation could be based on the different binding energies of water to the individual molecules. This may lead to different clustering in the expansions and consequently to different sizes of the formed neutral clusters, or to different decomposition after the interaction with the electron. However, the similarity of the mass spectra after EI indicates that there are no significant differences for the neutral precursor size and binding. This is supported by calculated binding energies of water for anion clusters with a different number of attached water molecules, Table 3. 12


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Figure 4: Distributions of the total excess energy released after electron attachment to uracils at different level of hydration as calculated in the present work. Thermal corrections were applied. While the values of the calculated binding energies are different for particular cluster sizes for different molecules, the mean values are similar for all three molecules. The specific fragmentation upon the EA is therefore caused mainly by the different energy transfer in the three systems. To estimate the energy transferred to the solvent, we compare our experimental data with ab initio calculations. Theoretical modeling of the electron attachment to nucleobases is generally difficult due to a delocalized nature of the attached electron, problems with self-interaction error and the need to treat electron correlation. 44–46 It has been shown that attachment to both isolated and hydrated nucleobases is driven by large dipole moment

13



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Table 3: Values of the binding energies in eV for binding of nth − water in the − − − X(H2 O)n− anion calculated as Eb (n− ) =E(X(H2 O)n− )-E(X(H2 O)(n− −1) ) n−

Eb U Eb FU

1 2 3 4 5 6 7 mean

0.72 0.37 0.85 0.63 0.71 0.61 0.51 0.63

0.72 0.6 0.61 0.61 0.47 0.79 0.53 0.62

Eb BrU 0.68 0.42 0.81 0.67 0.62 0.61 0.53 0.62

of these molecules e.g. 47–50 For isolated nucleobases, the dipole-bound state directly dissociates. 38,39 For solvated nucleobases, the dipole-bound state is supposed to transform into valence bound anion. 51–54 Full description of the attachment process then requires proper scattering calculation, 42,43 making the treatment impractical for solvated systems. The fact that the experiment was performed at single energy of the incoming electron significantly simplifies the theoretical modeling. The estimate of TET then does not require the value of vertical electron affinity but is obtained as a sum of the electrons initial kinetic energy of 1.2 eV and AEA (see eq. 2). Distributions of the TET values for the clusters with 0, 3 and 5 attached water molecules are shown in Figure 4. We observe an increase in the TET from U to BrU as well as a rise in the TET with the increasing level of hydration. Now we can explore, if the calculated energy transferred to solvent explains the differences between the individual molecules observed in the experiment. The TET values can be used to estimate the maximum number of evaporated water molecules for a particular microhydrated anion. For this estimate, we divided the mean values of the TET by the mean values of water binding energies from Table 3. Adding the number of evaporated water molecules estimated in this way to the experimentally observed mean value of waters attached to the anion, we can estimate the upper bound of the mean value max for the number of water molecules in the neutral clusters X(H2 O)n created in the expansion. The estimated values, together with the mean value of and 14


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= EI experiment

= EA experiment

max = + theory 10

Attached Water Molecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Higher Hydration (2bar)

Lower Hydration (1.5 bar)

1

U

FU

BrU

U

FU

BrU

Molecule X

Figure 5: Mean number of water molecules attached to U, FU and BrU cations after EI and anions after EA, as observed in the experiment. Mean value of the attached water molecules in neutral precursor max estimated on the basis of the hypothesis that all energy released in EA process results in the water evaporation. Lower and higher hydration conditions corresponds to experiments at expansion pressure 1.5 bar and 2 bar respectively. observed for the EI and EA are plotted in Fig. 5. Assuming that similar amount of energy is dissipated into water during the EI for all three molecules, the max for neutrals and for EI are expected to be parallel. This is roughly the case in Fig.5. This confirms our assumption about similar energy transfer to the solvent after EI as well as the fact that the simulations can qualitatively reproduce the observed differences for energy transfer to the solvent after the EA. A closer inspection of the numbers reveals small quantitative discrepancies for BrU clusters, which seem to lose more water molecules than we estimated based on the calculations, e.g. after the EI observed values are 0.7, 0.6 and 0.7 while the estimated neutral max values are, 6.2, 5.8 and 5.2 for U, FU and BrU, respectively. The most probable reason is the neglecting of the Br− signal channel in the estimate of the for the EA spectra. The clusters in which the molecule dissociates via Br− release can carry several water molecules. Even though the importance of this reaction channel decreases with the cluster size, its contribution can still increase the value of . Molecular dissociation (observed only for BrU) can also change the energy balance. Our calculation is based on the assumption that the energy transfer after the electron attachment to microhydrated molecules is equal to the energy required for the stabilization of the anion. 15



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For higher level of hydration, the cluster can trap both the Br− anion and Uracil-5-yl. radical dissociation products, while experimentally we observe BrU(H2 O)− n− anion clusters as well. As we already discussed, the calculations for bulk water 18 show that this process can add ∼0.3 eV energy to the system which may result in more effective evaporation of another water molecule. Finally, Figure 4 shows how the calculated TET distributions for different molecules change with hydration. We can see that for higher levels of hydration the distributions become wider due to the larger number of initial neutral configurations. For 5 water molecules the variance is already comparable to the mean value. Due to its resonant character, DEA can be selective to only one part of the distribution or only one neutral precursor structure. This possibility is also discussed in the recent theoretical work of Sieradzka. 43 Attachment to only one cluster structure corresponding to only one TET value out of the distribution may result in experimentally observed differences that do not correlate with the mean TET values. Exact scattering calculation is, however, beyond the scope of the present study.

Energy transfer to solvent after electron ionization Fragmentation of uracil and its substitutes after ionization in different environments has been extensively studied previously. 55–62 Therefore, we will focus here purely on the cationic clusters of the type X(H2 O)+ n+ , containing a parent molecule, which can give us new information about the energy transferred to the solvent. It is interesting that the degree of evaporation of water induced by 1.2 eV electrons and 70 eV electrons can be comparable. We can see e.g. on the top of Fig. 3 that after the ionization by 70 eV electrons evaporation of water molecules results in BrU(H2 O)+ n+ cluster distribution with ∼ 0.72 and the largest observed hydrated cluster with n+ =7, while the electron attachment to the same clusters results in BrU(H2 O)− n− cluster distribution with ∼ 1.1 and the largest observed hydrated cluster with n− = 4. After the EI in the bulk or large clusters, several processes can release energy that can be transferred to the solvent (see e.g. our works [ 63,64]). Most of 16


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these processes will result in the molecular fragmentation or release of much larger energy than estimated from the comparison of EA and EI MS in the present study (∼2.5 eV). The process responsible for the formation of non-fragmented clusters and water evaporation in the present experiment is therefore primarily the stabilization of a vertically ionized cation in its ground electronic state.

Relevance for radiotherapy The interaction of ionizing radiation with a living tissue produces a large amount of secondary electrons. 65 For example ∼104 secondary electrons are formed per each 1 MeV of energy and each high energy proton in water. 65,66 Let us assume that all these electrons thermalize to form stable anions and release several eV of energy per electron to water, as observed in our study. Then one would end up with an enhancement of the macroscopic linear energy transfer (LET) to the system in order of tens of keV per 1 MeV, which means few percents. This energy is deposited in an extremely short time and in an extremely small volume around the primary ion track. Such local heating can lead to a formation of shock waves and effective physical damage in the system as well as a long-range transport of the reactive species over the system. 67,68 The values of the energy needed to create such shocks are in the range of tenths of eV/atom, 69 which are similar to the values obtained in our studies of interaction of microhydrated uracils with ballistic electrons in vacuum. The situation in a real environment can be different. First, after irradiation, most of the electrons become solvated before they can reach the biomolecule. Second, the full water environment can change the reorganization energy in comparison to the simple model clusters. We try to estimate these effects for the studied molecules. We consider a hydrated electron in thermal equilibrium which is characterized by a hydration free energy of about -1.5 eV. 70,71 The reaction energy is then given as

∆Gr = Gaq (A− ) − Gaq (A) − ∆Gsolv (e− ) 17


(4)


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The energies of the neutral and anionic species were calculated again with the BMK/631++g** method, using the polarizable continuum model to mimic the dielectric continuum. The maximum values of the energy transferred to the solvent after interaction of solvated electrons with U, FU and BrU estimated from eq. (4) are 0.5, 0.2 and 0.8 eV respectively. These values are much smaller than the values calculated for microhydrated molecules. Surprisingly, in bulk water the excess energy for the FU molecule is lower than that of U and consequently, the highest difference in the released energy is between FU and BrU. Despite that, the difference is only 0.6 eV. This might not be enough to describe the known difference in the radiosensitizing activity of FU and BrU. Still, the energy transfer to the solvent may be an important contribution to the processes occurring during radiation interaction with living matter, especially concerning the secondary low-energy electrons. The importance may rise in systems containing components with high electron affinity such as gold nanoparticles. 72

Conclusions We present combined experimental and theoretical study of microhydrated uracils. We estimated how the interaction of secondary low energy electrons with uracils can enhance the linear energy transfer during high energy irradiation. We show that anion stabilization upon electron attachment leads to a significant energy transfer between the U, FU, BrU and neighboring water molecules. The calculations describe well the observed trends; however, they seem to underestimate the energy transferred in the case of BrU. This can be described by the dissociation of BrU and indirectly confirms the previous works describing the mechanism of BrU radiosensitization (e.g. [17,18]). However, the neutral configuration selectivity of the EA can also explain the observed differences in the energy transfer. It is interesting to note that the evaporation of water after the EA and EI to microhydrated BrU are similar. This indicates a similar energy transferred to the solvent for both processes. The similar energy transferred to the solvent after the EI for all three studied molecules then indicates that it is the result of the stabilization of a vertically ionized cation 18


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of the uracils.

Supporting Information Available Cartesian coordinates of the various optimized structures calculated in this work.

This

material is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgement This work has been supported by the Czech Science Foundation grant number 16-10995Y. MF, PS and JP thank to the Czech Science Foundation, grant number 17-04068S.

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