Microhydration Prevents Fragmentation of Uracil and Thymine by Low

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Microhydration Prevents Fragmentation of Uracil and Thymine by Low-Energy Electrons J. Kočišek,* A. Pysanenko, M. Fárník, and J. Fedor

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J. Heyrovský Institute of Physical Chemistry v.v.i., The Czech Academy of Sciences, Dolejškova 3, 18223 Prague, Czech Republic ABSTRACT: When ionizing radiation passes biological matter, a large number of secondary electrons with very low energies ( 0). This can be caused by better clustering of thymine with water (prior to the electron attachment) or by more effective evaporative cooling of the hot thymine anion−water complex after the electron attachment. The parent nucleobase anions have been also observed after electron interaction with nucleobase clusters in superfluid helium droplets.28 On the other hand, the same experiments with only single nucleobases in He droplets do not show any evidence for parent anions.29 In He, hydrogen abstraction becomes the most intense reaction channel, in strong contrast to present results for water. This difference is important in the context of biologically related studies because it shows that model rare gas solvent can not fully reproduce the effects caused by a more realistic solvent such as water. To our knowledge the only previous experimental work on electron attachment to hydrated a biomolecular model compound is the recent study of pyrimidine−water clusters.21 In contrast to the present systems, the (Pyr−H)− channel has been observed in ref 21 as the only molecular fragmentation channel after hydration, albeit shifted to higher electron energies when compared to isolated molecule. However, in isolated pyrimidine, this fragmentation channel is mediated by a core-excited resonance, occurs at much higher electron energies (around 5 eV), and has actually minor abundance when compared to fragments resulting from the ring-opening (that are quenched upon clustering). Also, in contrast to the present single microhydrated biomolecules, the target beam in ref 21 was a mixture of pure pyrimidine clusters, hydrated clusters, and pure water clusters, the pyrimidine−pyrimidine interaction could thus prevail over the pyrimidine−water interaction. A similar effect has been recently observed by us in hydrated nitrophenol clusters.22 How do the present findings influence the picture of the electron-induced damage of living matter? Initially, it has been suggested30 that process 1 produces H-radicals that eventually cause strand breaks. The occurrence of such direct fragmentation of nucleobases (especially thymine and guanine) even in solution by prehydrated electrons was later supported by the ultrafast spectroscopy data.6 Our results suggest that in water environment the anions are quickly stabilized and form bound states. (Here, one should distinguish between the ballistic electrons in the present work and prehydrated electrons in ref 6.) Such stabilization is an important part of a different, widely accepted model31−34 that the nucleobase serves as an “electron antenna” and captures the electron, which

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EXPERIMETNAL SECTION The experimental configuration (Figure 3) is identical to that described recently,22,40 apart from the molecular beam source.

Figure 3. Schema of the experimental setup.

The principle is as follows: the mixture of humidified helium and nucleobase vapor is expanded into the vacuum; the molecular beam is skimmed and crossed by an electron beam with a variable energy, and the negatively charged products are mass-analyzed by a reflectron time-of-flight mass spectrometer. The humidification of helium is achieved using a Pergo gas humidifier, a device commercially available from Elemental Scientific, originally intended for humidification of argon in inductively coupled-plasma mass spectrometry. It utilizes Nafion tubing that selectively permeates water vapor through its membrane, which humidifies the helium line. The 3403

DOI: 10.1021/acs.jpclett.6b01601 J. Phys. Chem. Lett. 2016, 7, 3401−3405

Letter

The Journal of Physical Chemistry Letters humidified buffer gas then passes through an oven filled with uracil or thymine powder, which is connected to a conical nozzle with 90 μm diameter. The oven reservoir and the nozzle are both separately temperature-controlled; the latter is kept at slightly higher temperature in order to prevent sample condensation. The expansion conditions are summarized in Table 1. The beam is skimmed and passes three differentially

attachment. If the binding energy of one water molecule to the complex is 0.3 eV,41 the excess energy is sufficient to evaporate six H2O molecules. However, the number of evaporated molecules will certainly be lower because considerable excess energy will be carried away as the kinetic energy of evaporating units. The target beam is thus indeed composed of individual microhydrated nucleobase molecules.

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Table 1. Conditions in the Molecular Beam Sourcea sample species uracil thymine hydrated hydrated hydrated hydrated

uracil I thymine I uracil II thymine II

P0 [bar]

Tr [K]

Tn [K]

Tw [K]

1.5 1.5 0.8 0.8 1.5 1.5

463 468 458 458 458 458

463 473 463 468 463 468

− − 301 301 301 301

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the Czech Science Foundation Grant 16-10995Y.

a

P0, Tr, Tn, and Tw represent stagnation pressure and temperatures of the nucleobase reservoir, nozzle, and water reservoir, respectively.

REFERENCES

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pumped vacuum chambers where it can be probed by other techniques not used in this work. The interaction point with the electron beam is thus located 1.5 m downstream from the nozzle. The electron beam is produced in a pulsed electron gun (pulsing frequency 10 kHz). The electrons pass the interaction region for 2 μs, and after a 0.5 μs delay, a 2 μs long negative high-voltage pulse is applied to extract the created anions to a reflectron time-of-flight mass spectrometer (RTOF). The mass resolution of the RTOF is 5 × 103. The electron-energy scale is calibrated using the 2.2 eV band in the O− ion yield from N2O. The energy spread of the electron beam is approximately 600 meV. Test experiments with SF6 revealed that below 1.3 eV the electron current passing the interaction region with target gas quickly drops, and the current recorded in the Faraday cup does not correspond to the actual current in the interaction region. This is caused by the construction of the electron gun which was optimized for operation at energies of several tens of electronvolts. The present ion yields are thus reliable at energies above 1.3 eV. Composition of the Beam. Both positive and negative ionization can induce fragmentation of the weakly bound species; the unambiguous determination of the beam composition is thus not possible using mass spectrometric techniques. However, two observations can be immediately made from the negative ion mass spectra of the hydrated beams (Figure 1): (i) no U or T dimer anions (neither pure nor hydrated) are observed and (ii) the hydration conditions I clearly result in much smaller NB(H2O)−n anions than the hydration conditions II. Observation (i) shows that the partial pressures are set such that the adiabatic cooling in the expansion leads largely to the nucleobase−water clustering. The cooling (resulting from the collisions with helium in the expanding gas flow) is of course more efficient if the higher stagnation pressure is used. Hence, larger nucleobase−water complexes are produced at conditions II, still without evidence for the formation of pure nucleobase clusters. The production of the smallest anions NB(H2O)n−, n = 0, 1, 2 at conditions I suggests that the number of water molecules around the nucleobase is in the order of units, just from the energetical reasons. Taking the electron−complex collision energy of 1.5 eV and adiabatic electron affinity of hydrated anions 0.5 eV12 indicates 2 eV is available for evaporation of water monomers in the course of electron 3404

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