Study of complex molecules of biological interest with synchrotron radiation

Accepted Manuscript Title: Study of complex molecules of biological interest with synchrotron radiation Author: K.C. Prince P. Bolognesi V. Feyer O. Plekan L. Avaldi PII: DOI: Reference:

S0368-2048(15)00193-0 http://dx.doi.org/doi:10.1016/j.elspec.2015.08.010 ELSPEC 46494

To appear in:

Journal of Electron Spectroscopy and Related Phenomena

Received date: Revised date: Accepted date:

25-2-2015 10-8-2015 17-8-2015

Please cite this article as: K.C. Prince, P. Bolognesi, V. Feyer, O. Plekan, L. Avaldi, Study of complex molecules of biological interest with synchrotron radiation, Journal of Electron Spectroscopy and Related Phenomena (2015), http://dx.doi.org/10.1016/j.elspec.2015.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Study of complex molecules of biological interest with synchrotron radiation

K. C. Princea, b, c, P. Bolognesid, V. Feyera,e, O. Plekana and L. Avaldid

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a) Elettra-Sincrotrone Trieste, Strada Statale 14 - km 163,5 in AREA Science Park, I-34149 Trieste, Italy b) Istituto Officina dei Materiali, Consiglio Nazionale delle Ricerche, in Area Science Park, I-34149 Trieste, Italy, c) Molecular Model Discovery Laboratory, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Melbourne, Victoria, 3122, Australia d) CNR-ISM, Area della Ricerca di Roma 1, Via Salaria Km. 29,300, Monterotondo (Roma), Italy e) Present address: Forschungszentrum Jülich GmbH - IFF - IEE, 52425 Jülich, Germany

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ABSTRACT

Synchrotron radiation and synchrotron based spectroscopic techniques have found important

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applications in the study of isolated molecular species of biological interest. In this paper some examples of spectroscopic and dynamic studies of amino acids and small peptides, nucleobases and pharmaceuticals are

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reviewed. Opportunities offered by the advent of new radiation sources combined with novel methods for

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1. Introduction

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the production of beams of these molecules are also discussed.

The macroscopic effects induced in living cells by the absorption of ionizing radiation are closely

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related to the structural and chemical properties of their molecular constituents and can be related to events on a microscopic scale, with processes initiated at the atomic and molecular level. It is well known, for example, that substantial damage to DNA/RNA can be produced by slow electrons with energy of a few eV [1], produced either directly or via ionization of a medium, and this has been widely investigated. Moreover, understanding of the physics and chemistry of isolated molecules of biological interest is fundamental not only for biology, but in biotechnological applications, such as sensors and molecular electronics, and in astrochemistry and astrobiology, where for example key information on the origin of life in the universe is provided by the understanding of the chemistry of relatively simple molecules in an environment subject to ionizing radiation. In this multidisciplinary field, atomic and molecular physics can provide a valuable contribution both experimentally and theoretically. Gas phase studies enable the distinction between intrinsic properties of the molecules and those due to the interaction with the environment [2-6] and therefore provide important benchmark data for studies of liquid solutions or in the solid state. Moreover, studies of isolated biomolecules can benefit from all the armoury of theoretical techniques developed for polyatomic molecules, such as ab-initio calculations and DFT methods. 1 Page 1 of 23

Synchrotron radiation with its tunability over a broad energy spectrum and synchrotron based spectroscopic techniques represent a unique combination to investigate the absorption of a defined amount of energy by a molecule, sometimes even at a specific bond, and then to probe the effects of this excitation on the electronic structure and stability against fragmentation of the molecule. Knowledge of the electronic structure and spectroscopy of these molecules is of fundamental importance for understanding

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their functioning and possible malfunctioning.

Recently, several groups worldwide have successfully tackled different aspects of some classes of

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molecules of biological interest. For example photoelectron spectroscopy has been used to examine the relation of valence and inner shell spectra to the shape of amino acids [2,7] . Amino acids generally exist at

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laboratory temperatures as several conformers of comparable energy, and the determination of their properties is of great importance. The selectivity of inner shell ionization of carbon, nitrogen, and oxygen atoms of DNA/RNA molecules [8] has been correlated with double strand breaks and the inactivation of

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various cells. A combination of techniques including inner shell absorption, X-ray photoemission, mass spectrometry [3-10] has been used to develop an understanding of the radiation damage to DNA/RNA and their building blocks and to disentangle the effects induced directly on the isolated molecule itself from the

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indirect ones induced by the surrounding environment. More sophisticated approaches based on coincidence techniques [10, 11] have allowed the chain of processes after the energy absorption to be

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followed.

One of the main challenges in the field is the production of a target beam with a suitable density of

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molecules. Most of these samples are solid at room temperature, and one method is to evaporate them from an appropriate furnace, taking care to avoid thermal decomposition, but this has set some limitations

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on the classes of molecules studied. For example, while a broad literature exists on amino acids and nucleic acids, studies of the nucleosides and nucleotides are scarce, because these larger molecules are more thermally sensitive.

The paper is organized as follows. The next section is devoted to the methods used in the studies of

molecules of biological interest, focusing on the experimental methods. In section 3 some examples of spectroscopic and dynamic studies of amino acids and small peptides, nucleobases and pharmaceuticals are reported briefly. Finally section 4 is devoted to some perspectives triggered by the advent of new radiation sources and conclusions.

2. Methods The investigation of gas phase biomolecules poses new challenges compared with the investigation of smaller and more volatile molecules. Most biomolecules, including even the smallest such as nucleobases and amino acids, are solid at room temperature, and as stated, have to be brought into the gas 2 Page 2 of 23

phase to be studied at the single molecule level. In the case of DNA/RNA bases a solid ring structure makes these molecules (except for guanine) quite resistant to thermal decomposition, so that they can be evaporated from ovens to produce effusive or supersonic beams of neutral molecules. In other cases, e.g. some nucleosides and amino acids with reactive side chains, great care has to be taken to characterise the working conditions that guarantee the evaporation of intact molecules. For most of the more complex

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systems, thermal evaporation cannot be employed, due to the fragility of the targets. In such cases, alternative and more elaborated approaches like Electrospray Ionisation (ESI) or Matrix Assisted Laser

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Desorption Ionisation (MALDI) can be used instead of thermal evaporation. However, protonated and multiply charged species are produced by these methods, and the sample density is low. The approaches to

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produce the target beam are briefly described in section 2.1, while the typical synchrotron based spectroscopic techniques, with specific reference to the study of biomolecules, are briefly reviewed in

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section 2.2.

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2.1 The targets

The typical experimental set-up used for the evaporation of small biomolecules is composed of a resistively heated oven that heats a crucible containing powder of the target molecule. The heating is often

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provided by ceramic insulated wires or commercial Thermocoax heaters [12] providing up to several tens of

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Watts of power. The winding of the oven should be non-inductive to avoid spurious magnetic fields in the set-up. Furthermore, the top and the bottom of the crucible are normally held at slightly different

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temperatures, with the top being hotter than the bottom in order to prevent condensation and blockage of the narrow orifice at the exit of the crucible. The oven is contained in a high or, even better, ultra-high vacuum chamber, bakeable and equipped with a cold trap opposite the oven, to collect the vapour. The cold trap helps to maintain low background pressure, which is particularly important in mass spectrometric studies of biomolecules, where the relatively low density of the vapour beam requires low background pressure in order to produce a good signal-to-noise ratio. Efficient trapping is necessary to avoid deposition of an insulating layer on the electrodes of the electron and ion analysers, which can seriously affect their efficiency and stability. Last but not least, baking the entire vacuum system is an efficient way to clean the set-up of residual contamination; this is a standard procedure for Ultra High Vacuum systems and it is best to provide this capability even for high vacuum systems. Design considerations for ovens have been published for evaporation of metal vapours [13], and similar ideas have been recently adapted and optimised for the evaporation of fragile biomolecules [14-19]. These more specific approaches are based on the use of non-metallic and more inert materials for the crucible, strict control of the working conditions and a careful characterisation of the thermal decomposition of the target molecules. 3 Page 3 of 23

Thermal evaporation has the advantage of producing a beam of neutral molecules. However, it has limitations for the study of relatively complex biomolecules, especially if they contain reactive side groups such as carboxylic acid, hydroxyl, and sulfhydryl. Novel methods of vaporising biomolecules are required

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and some promising techniques will be discussed in section 4.

2.2 Analytical methods

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Synchrotron radiation is the most powerful radiation source in terms of the broad range of tunability of the photon beam, continuous from the UV to hard X-rays. This allows for photoionisation and

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photoexcitation experiments, able to map out both occupied and empty states respectively, of the valence as well as in the inner shell orbitals of the target. Photo Emission and X-ray Photoemission Spectroscopies (PES and XPS), where the kinetic energy (KE) of the photoionised electron is measured at a fixed photon

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energy (hv), allows reconstruction of the electronic distribution of the molecular orbitals of binding energy BE = hv - KE. In the valence shell, especially from comparison with theoretical predictions, important information about the electronic charge distribution of the orbitals in the molecule can be derived. In the

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inner shell, the localised nature of the core electrons implies that each atom is affected by its surrounding chemical environment and site-selective information can be obtained: use of the binding energy shifts to

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obtain chemical information is also known as ESCA, Electron Spectroscopy for Chemical Analysis. Subtle differences among families of similar molecules (e.g. isomers, or analogues), or the effect of

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functionalisation can be identified, assessed and discussed in terms of the measured and calculated innershell chemical shifts. Electron energy distributions measured over a broad range of kinetic energies from

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zero up to hv can be used to characterise the complete electron emission spectrum due to the photoemission as well as to the Auger decay of core ionic states, or autoionisation of excited states. In Fig. 1 for example, the C 1s spectra of the three related molecules caffeine, xanthine and hypoxanthine are shown [20]. These three molecules are all purine derivatives, and caffeine is formed from xanthine by the addition of three methyl groups. The spectrum of caffeine is not simply that of xanthine with the addition of a peak due to the three extra carbon atoms, but is also shifted in energy, as predicted by theoretical calculations. Hypoxanthine is formed from xanthine by subtraction of an oxygen atom, but the spectra show that at least two different forms (tautomers) are present in the gas phase.

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Fig. 1 Experimental (points) and theoretical (thin lines) C 1s spectra of caffeine, xanthine and hypoxanthine [adapted from ref. 20].

Complementary information to that provided by electron emission, which probes the occupied

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states, can be obtained by probing the empty states. Near Edge X-ray Absorption Fine Structure (NEXAFS) provides electronic structure data, promoting core electrons to empty (excited) states. The experiments

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can be performed by scanning the photon energy across an inner shell threshold and measuring a signal that is proportional to the absorption cross-section, for example the total ion yield. In biomolecules, C, N, O

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and S are typically the atoms that can be probed in the soft X-ray region, near their K-edges [21]. A simple method of measuring the NEXAFS spectrum is to use an ion detector consisting of a channel electron

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multiplier (or channel plate), and to measure the current, or number of ions (pulse counting). Fig. 2 shows the nitrogen K edge spectrum of glycyl-glycine, the simplest peptide, compared to theory.

Fig. 2 Experimental (red line) and theoretical (black line, and sticks) nitrogen K edge spectra of glycylglycine. The inset shows an enlargement of the first part of the spectrum, compared to the monomer glycine (green curve) [21]. 5 Page 5 of 23

If the electron spectrum is measured with a photon energy corresponding to a core resonance, previously determined using NEXAFS, the technique is known as Resonant Auger spectroscopy. It differs from normal Auger spectroscopy because it involves the decay of a neutral excited state to a singly charged ionic state, whereas the normal Auger process is the decay of a singly charged ion to a doubly charged ion. Similarly, autoionisation photoelectron spectra can be measured: they are usually due to excitation to a

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resonant valence excited state, which subsequently decays by emitting an electron. To know the energy of the resonance, a scan in energy is performed at low energies, similar to a NEXAFS scan near the inner shell

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threshold. The full electron emission spectra, including photoelectron, Autoionisation, Auger and Resonant Auger electron characterised at different photon energies are useful benchmark data for Monte Carlo and

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Ion Tracking simulation codes, used to evaluate the direct and indirect radiation damage due to the interaction of ionising radiation with a biological medium. The valence and core spectra are also useful as benchmark data for calculations of molecular properties, which start from a calculation of the electronic

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structure, particularly the valence electronic structure. If the calculation can reproduce accurately the measured valence and core spectra, and also the geometric structure (usually the calculated structure is compared with crystallographic data), then it is reasonable to expect that other calculated properties are

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accurately predicted, for example dipole and multiple momenta, appearance energies, electron and proton affinities, reaction rates, etc.

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If ion detection is performed using a technique that disperses the fragments according to their mass, then even more information is obtained, and the technique is known as photoionization mass

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spectrometry (PIMS). The most common detectors are time of flight (TOF) [22] or quadrupole mass spectrometers (QMS). With these detectors, the NEXAFS spectrum can be recovered by scanning the

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photon energy, and summing the signal at each energy. As well, the fragmentation pattern of the molecules is obtained at each photon energy, and it is possible to relate the UV or soft x-ray photon energy absorbed to the products of dissociation. For valence band ionization, the experimental spectrum can be compared to a calculated spectrum, and from this it is known which orbitals are ionized. The highest kinetic energy electrons leave the molecule in its ground ionic state, which may or may not be stable. All other ionic states are excited states, and since they contain more energy than the ground ionic state, they have a greater tendency to fragment. As the photon energy increases, more of these excited ionic states are produced, and this generally leads to stronger dissociation. In the soft x-ray region, excitation of core level resonances gives rise to the Resonant Auger spectrum for electrons, but it is not so simple for predicting the ionic fragments produced. The situation is complex, because even if the excitation is localised, after electronic decay (on the scale of a few fs), an excited state may be produced in which energy flow through the molecule occurs, before fragmentation, which may occur on the ps or longer time scale. For ionization, rather than excitation of a core level, the core hole decays via an Auger process, again on the scale of a few fs, and generally produces a doubly 6 Page 6 of 23

charged ionic state, with both holes in the valence band (KVV decay). The fragmentation is then very rapid (if the molecule is not too large) as there is a strong Coulomb repulsion between the two holes. However, this situation is also rather complicated, as there are very many two hole states, and it may be difficult to identify these. The simultaneous detection of electrons and ions in coincidence, that is from the same ionization

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event, adds further insights into molecular fragmentation. In photoelectron-photoion [23] and photoelectron-photoion-photoion coincidence (PEPICO and PEPIPICO) [24] experiments, the ionization rate

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is reduced to a sufficiently low value that in a given time interval (the time resolution of the detection system), there is likely to be only one ionization event. The electron kinetic energy determines which

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valence or inner shell orbital has been ionized, providing state- and site-selectivity (for valence and core ionization respectively), while the measurement of the mass spectrum in coincidence gives information about the molecular fragmentation of that specific state. In the PEPIPICO experiments, also the detailed

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description of the dissociation process of doubly/multiply charged ions and determination of kinetic energy released in the process (available from TOF spectrometers) can be achieved providing a very comprehensive picture of the dynamics of fragmentation. Fig. 3 shows the PEPICO spectrum of pyrimidine.

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The ion at mass 80 is the intact molecule, and ionization of the first few molecular orbitals produces this ion, but ionization of deeper orbitals does not produce this ion, that is, the molecule always fragments. The

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fragment at mass 53 is produced by ionization of orbitals from about 12.5 to 15 eV, while more highly

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excited ionic states give rise to the smaller fragment at mass 26.

Fig. 3 Upper panel: valence band photoemission spectrum of pyrimidine. Lower panel: photoemission spectra in coincidence with selected masses. Photon energy is 21.2 eV [23]. 7 Page 7 of 23

3. Spectroscopy and dynamics with synchrotron radiation 3.1 Amino acids and small peptides. An important class of biomolecules consists of amino acids, their derivatives, and their oligomers, that is, peptides [2,7,21-31]. They are built from three or four motifs: a carboxylic acid group, an amino

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group, a side chain, and for peptides, a peptide group. The amino acids and linear peptides are generally “floppy” molecules, that is, rotations around bonds have low energy barriers and many conformers are populated at laboratory temperatures. The energetic ordering of the conformers is usually governed by

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hydrogen bonds with the amino and carboxylic groups acting as donors or acceptors, depending on the conformation. A particularly clear manifestation of how this is visible in photoemission spectroscopy is the

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case of proline, where two pairs of conformers are populated, with the amino nitrogen atom acting as a

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hydrogen donor or acceptor, Fig. 4 [2].

Fig. 4 Experimental N 1s photoemission spectrum of proline at two temperatures, and the calculated spectrum (lower curve) [2].The agreement for the energies of the peaks is good, but the intensities do not agree, indicating that the populations are not correctly predicted. The inset shows the lowest binding energy part of the valence band at the two temperatures. In Fig. 4, the core levels of nitrogen are clearly split into two components. Since proline contains

only one nitrogen atom, it is immediately clear that there are at least two different chemical states present, that is, two conformers. Calculations and measurements of the conformer structures [32] show that there are in fact four, and that these belong to two groups: those with the amino group acting as a donor, and those with it acting as an acceptor. An advantage of the use of core level spectroscopy is that it can be performed at variable temperature, whereas a number of other spectroscopies require a cold molecular beam. A second advantage of photoemission is that relative intensities are substantially quantitative, whereas methods such as infrared, laser and microwave spectroscopy usually require a calculation of transition probabilities in order to quantify the relative intensities in a spectrum. The quantitative nature of the photoemission spectra has been applied to extract thermodynamic parameters [28]. 8 Page 8 of 23

We note that the method of vaporising the sample may influence the observed spectrum. In particular, thermal evaporation produces molecules that are vibrationally excited, that is, they contain internal energy, whereas other methods such as MALDI and electrospray ionization produce cooler molecules. In valence and core spectra, this produces “hot bands” which may broaden the spectra. In the case of core level spectra, the measurement remains quantitative as the same integrated intensity is

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produced. Generally the temperatures used are moderate, and so the additional broadening with respect to 300 K is small. Close to threshold, the first ionization potential may appear to be lowered, because the

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sum of the internal and photon energy may be sufficient to ionize the molecule by an autoionization process [33], so that thermal effects may become important in high precision photoelectron spectroscopy.

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For ion spectroscopies, the internal vibrational energy adds to electronic energy, and may significantly increase fragmentation.

More complicated molecules such as peptides can also be probed [21]. Linear peptides contain a

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single peptide bond and in the simplest molecule of this class, glycyl-glycine, it is possible to probe the electronic structure in detail [21]. For example, both the valence band and core electronic structures are closely related to, but distinct from, glycine.

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Peptides can also be cyclic, and form rings bound by peptide bonds, and many of these cyclic peptides are biologically active. The simplest cyclic peptides consist of two peptides, and the centre ring

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with two peptide bonds is also known as diketopiperazine; the amino side chains are then attached to this moiety, fig. 5. Linear peptides have an amino group at one end and a carboxylic acid group at the other, but

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because of their absence in cyclic peptides, these compounds are more stable, for instance resisting

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digestion in biological systems, and resisting thermal decomposition when evaporated in the laboratory.

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Fig. 5 Structures of three cyclopeptides: (a) cyclo (Glycyl-Glycyl), (b) cyclo (Leucyl-Prolyl) and (c) cyclo (PhenylalanylProlyl) [30].

3.2. Nucleobases. There are five DNA/RNA nucleobases: guanine, adenine (both are derivatives of purine), cytosine, thymine and uracil (derivatives of pyrimidine). These molecules, their parent compounds and many derivatives and analogues have been studied by synchrotron radiation and related techniques; a selection of our work is given in refs. [3, 34-40], investigations by other groups of the valence electronic structure by photoemission 9 Page 9 of 23

can be found in refs. [41-48] and studies of dissociation and fragmentation following VUV and soft X-ray absorption are reported in refs [11, 19, 41, 49-56]. A notable feature of guanine and cytosine is that in the gas phase at laboratory temperatures, there are several tautomers, that is, isomers in which the heavy atoms have the same arrangement, but the hydrogen atoms and some single and double bonds have different positions. In solution, which is the state of greatest biological interest, the tautomeric equilibrium by solvent effects and it is more difficult to determine intrinsic properties.

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is different, so different populations are observed. However, in this state spectroscopic data are influenced

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Figs. 6a-d show an example of pyrimidine and some of its halo substituted derivatives, demonstrating how the photoemission technique can be used to obtain insights into the molecular

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nN+

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structure.

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3

1

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nN-

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Fig. 6 a-d) The pyrimidine, 2-bromo, 5-bromo and 2-chloro pyrimidine molecules. e) The He(I) PES spectrum of pyrimidine and f) some of Hartree-Fock molecular orbitals respectively measured and calculated in ref. [57]. The asterisk in the PES spectrum marks the peak due to ionization of water molecules in the sample.

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The halogenated pyrimidines constitute an important class of prototype radiosensitising molecules, and therefore the study of their properties is of interest to understand the fundamental mechanisms of the enhanced radiation damage when these molecules are selectively incorporated into the DNA of tumour cells. The first photoionisation experiments in pyrimidine [47-48] and halogenated pyrimidines [57] were performed in the valence shell using synchrotron radiation or the fixed wavelength of a He(I) discharge

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lamp. The comparison of the PES spectra with the theoretical calculations is an essential step in the assignment of the features to specific molecular orbitals (see Figs. 6e and f) and to discuss the substituent

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effects of the halogenation (see Fig. 6a-d).

The halogen atom affects the energies of the  orbitals of the pyrimidine ring via two, often

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antagonistic mechanisms, the resonant (R) and inductive (I) effects. The I effect is due to the electronegativity of the halogen substituent which withdraws electron density from the ring thus increasing the binding energy of the orbital. The R effect is due to conjugation between the lone pair orbitals of the

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halogen atom and the  orbitals of the ring. The effect depends on the conjugation, and can be either stabilising or destabilising, i.e. decreasing or increasing the binding energies, respectively. The R and I

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effects are manifested in several molecular properties, from PES and XPS binding energies to molecular fragmentation. In the PES spectrum, the resulting sum of the two effects on the binding energy of the halogenated pyrimidines is shown in Fig. 7a. A similar interplay between R and I effects was also observed

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in the C 1s ionisation shell of these molecules [3] (see Fig. 7b). In this case, the site-selectivity of the inner shell ionisation allows further discussion of how each atom in the molecule contributes to the screening of

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the core vacancy. This information is very important for the interpretation of the mass spectra of the

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fragmentation of these molecules.

Fig. 7a The correlation diagram for the valence states of Fig. 7b The correlation diagram for the C 1s pyrimidine and the halogenated pyrimidines as calculated using the B3LYP method in ref. [57].

halogenated pyrimidines from the experimental measurements (in black) and DFT calculations (in red) as from ref. [3].

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A joint theoretical and experimental investigation of the appearance energy (AE) of these same halopyrimidines [9] has led to interesting conclusions related to the radiosensiting behaviour of these molecules. Castrovilli et al. [9] found that the HCN loss channel is favoured with respect to the halogen radical loss channel in the 5-bromopyrimidine case, whereas the opposite occurs in the case of the 2-chloro and 2-bromopyrimidine molecules. The 2(HCN) loss was observed only in the 5-bromopyrimidine. These

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findings suggested that the presence of the Br atom in position 5 favours the breaking of the ring, whereas the presence of the halogen in position 2 favours the release of the halogen or H radical, with release of the

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Br• radical in the brominated cases and of the H• radical in the chlorinated case. Similarly to the PES and XPS studies, these effects have been clearly pinpointed by the theoretical calculations and also rationalised,

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on a more qualitative level, in terms of the interplay of R and I effects, as well as of the different electronegativity of the Cl and Br halogen atoms. As a consequence, these results suggested not only the possible mechanisms of radiosensitisation of the halopyrimidine with respect to the unsubstituted

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pyrimidine molecule, but also that the choice of the most suitable radiosensitiser should be dictated by the goal of the therapy. 5-bromopyrimidine can induce more significant breaking of the ring, and therefore direct damage of the DNA where it has been selectively incorporated. On the other hand, the halogen atom

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in position 2 leads to the efficient release of Br• or H• radicals, which act on the environment surrounding the cell nucleus. All of these examples show that in both the valence and inner shell cases, the specific

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halogen substituent as well as its position on the ring play a very important role in the valence as well as inner shell electronic structure of these molecules, affecting the stability of the electronic states and, in

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turn, their fragmentation pattern.

As far as the fragmentation is concerned, an important question regards its site- and state-

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selectivity, i.e. the possibility to control and address the molecular fragmentation by a suitable choice of the photon energy. To answer this question, several mass spectrometric and PEPICO experiments have been performed both on the valence and inner shell regions of the halopyrimidines, where most spectroscopic characterisation is available. The PEPICO experiments showed that in pyrimidine [23, 10] and halopyrimidines [58] the different valence electronic states have a marked selectivity in the fragmentation. In general terms, it was shown that i) the intact, parent molecule is only observed in the ground electronic state and up to about the lowest fragment Appearance Energy; ii) for increasing internal energy, as new fragmentation channels open up, increasingly smaller fragments populate the mass spectrum, with the simultaneous disappearance or very significant reduction of the intensity of the larger fragments observed for the lower ionic states; iii) ionic states preferentially correlate to the nearest fragmentation threshold, even though lower fragmentation channels are energetically open [10, 58]. The observation of this strong state-selectivity raises the question whether the site-selectivity, i.e. the possibility that the specific site in the molecule where the energy has been deposited, also affects the molecular fragmentation. This question cannot be answered by the previous investigation, due to the 12 Page 12 of 23

delocalised nature of the valence orbitals. The inner shell, localised, electrons have to be considered. Siteselectivity similar to that in core ionisation can be achieved via resonant core excitation, by suitably selecting the appropriate resonant photon energy from the NEXAFS spectra [38]. With respect to core ionisation, the core excitation has the advantage that, just by choosing the correct resonant photon energy, it is possible to select the atomic site in the molecule where the energy has been deposited. Mass spectra

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measured at different photon energies on and off resonant excitation [10] around the different K-edges in the pyrimidine molecule show that the molecular fragmentation is very sensitive to the ‘localisation’ of the

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energy deposition in the different sites in the molecule. Indeed, not only the total ion yield is orders of magnitude more intense on resonance, but the fragmentation is also qualitatively different (see Fig. 8).

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These resonantly excited states decay predominantly by non-radiative processes to one-hole and two-holeone-particle final states, via so-called ‘participator’ and ‘spectator’ Resonant Auger emission (RAE), respectively. These states correspond to increasingly excited states of the singly charged ion. The RAE

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spectra resemble the valence photoelectron spectrum (PES), but with an intensity distribution that depends on the overlap of the wave functions of the intermediate (core excited) and final (valence ionic) states. In a RAE – ion coincidence experiment it is therefore possible to achieve complete control of the process, as the

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photon energy selects the atomic site of the energy deposition, the kinetic energy of the RAE selects the binding energy of the final ionic state populated by the electron decay and the mass spectrum measured in

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coincidence associates a fragmentation pattern to this specific process, providing simultaneously the siteand state- selectivity. Such an experiment performed on the pyrimidine molecule [10] has shown that the

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fragmentation does not depend on the intermediate, but only on the final singly charged ion state. In other words, as long as we fix the final state reached by the electron decay of the intermediate, inner shell

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excited state, it does not matter anymore whether the energy was initially deposited on the C or N atoms, as the molecular fragmentation follows the same pattern (see reference [10] for a detailed discussion). The electron decay is generally much faster than the molecular fragmentation. Therefore, one can expect that the direct or resonant ionisation will populate the valence shell orbitals of the intact molecule before fragmentation, and with different branching ratio. Considering the state-selectivity in the fragmentation of the ionic states, this explains the differences observed in the mass spectra of Fig. 8.

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Fig. 8 The TOF spectra of pyrimidine measured at the N(1s  π*) resonance, 398.8 eV photon energy (red) and in the

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ionization continuum below the resonance, at 385 eV (black). An overview of the full mass spectrum is shown in the bottom panel, while expanded views are reported in the upper panels, ref [10].

The RAE – ion coincidence experiments are an interesting proof of principle of the potential information that can be obtained about the structure and dynamics of ionisation of biomolecules, and how

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this can be related to specific questions of radiation damage. For the typical photon energies used in radiotherapy, the radiation damage is more likely to be triggered by ionisation, rather than excitation

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processes. A similar investigation can be performed for the decay of core ionic, rather than excited, states. However, considering that the dominant, non-radiative, decay in this case will be to doubly charged states,

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a complete description can be achieved only in photoelectron – ion – ion [51, 11] and Auger electron - ion – ion [52] coincidence experiments. In principle the photoelectron/Auger electron can be used to define the core vacancy/doubly charged state respectively, and the ion – ion- coincidence map will associate two correlated fragments. In practice, due to the complexity of the polyatomic targets and to the very large number of overlapping doubly charged states it is very difficult to reach good selectivity for the channel involved.

The study of isolated nucleobases provides information about intrinsic properties of these

fundamental building blocks of DNA/RNA. However, in order to approach more realistic conditions, it is essential to increase the size of the biomolecule, combining different elements that would naturally host the base in real DNA/RNA. This involves moving from isolated bases to nucleosides and nucleotides, including the bonding of the nitrogenous base with a ribose or ribose plus phosphate group, respectively. Due to the fragility of these molecules, very few synchrotron radiation studies have been presented of nucleosides in the gas phase [53-55], and none of the neutral nucleotides. The latter have been studied as charged species produced by the ESI technique [56]. 14 Page 14 of 23

3.3 Other biomolecules. Another field of application of synchrotron radiation is to pharmaceuticals and other molecules with biochemical roles, with a view to understanding their electronic properties, and modelling their chemical behaviour. This includes antibiotics and their building blocks, radiosensitizers for radiotherapy,

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but also sugars, lipids, and a range of other materials. Here we give just one example.

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Fig. 9 Outer valence band spectra of cycloserine and 2-oxazolidinone, showing the first 4 ionic states. Bottom curve: calculated energies of the first states. Upper curve: experimental spectrum. Insets: the calculated charge distributions of the first three states [59]. The relatively small molecule cycloserine is an antibiotic and its closely related analogue, 2-

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oxazolidinone, is the parent molecule of a number of potent antibiotics. Photoelectron spectra of the valence and core levels of this pair of compounds have been reported [59]. The outer valence band spectra of these two compounds show some similarities, but the charge distributions are different. These outer or frontier orbitals are the ones involved in chemical reactions and so govern the chemistry of their antibiotic activity.

In the case of radiosensitizers, i.e. drugs that are used in chemotherapy to enhance the effects of

radiation, synchrotron radiation can provide various kinds of information. On the one hand, its tunability allows selection of a photon energy close to the K-edge of an element in the radiosensitizer and therefore to enhance the photoelectric effect directly during therapy. Considering that among the most used drugs are cisplatin and carboplatin, iodinated contrast agents and iododeoxyuridine and motexafin gadolinium, keV photons must be used [60, 61]. On the other hand soft X-rays with their chemical sensitivity can be used to shed light on the basis of the synergic interaction between radiation and the drug. The case of isolated molecules like halopyrimidines has been already described in the section 3.2, while applications aim to disentangle the mechanisms proposed for the effect. In the case of cisplatin, one proposed 15 Page 15 of 23

mechanism attributes the synergistic effect to the inhibition of repair of radiation induced damage to DNA, while another one assumes that the species created by the primary radiation in cells cause additional damage when cisplatin is covalently bonded to DNA. A study of this kind has been published by Xiao et al [62].

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4. Perspectives and conclusions

In the previous sections, the possibilities offered by synchrotron radiation techniques to investigate

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the electronic structure, the conformation and the dynamics of fragmentation of molecules of biological interest have been discussed. While most of the work performed up to now and all the work done by the

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present authors has been restricted to thermal evaporation, and therefore to small molecules, a promising research area for the future is the examination of larger molecules, brought into the gas phase by sophisticated and softer methods. Alternative techniques to produce vapour beams of biomolecules are

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Matrix-Assisted Laser Desorption/Ionization (MALDI) [63] and Laser Induced Acoustic Desorption (LIAD) [64]. In MALDI the sample is mixed with a suitable matrix material on a metal plate. Then a pulsed laser

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irradiates the sample, triggering ablation and desorption of the sample and matrix material; typically the matrix strongly absorbs the laser light. In the LIAD technique a sample is deposited on the surface of a thin metal foil (e.g. tantalum) which is then back-irradiated, using a nanosecond (ns) UV pulse. This interaction

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stimulates the propagation of an acoustic wave through the foil, resulting in sample desorption from the

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surface. In contrast to MALDI, LIAD produces a pure, neutral target of biomolecules, and has been successfully applied to amino acids by Calvert et al. [65], with ionization by optical lasers. Both of these

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sources are pulsed and when used with a continuous source like the synchrotron radiation suffer from a low duty cycle, and at present none of these techniques has been coupled with synchrotron radiation. However it should be possible to do this as pulsed sources of other materials [66], with repetition rates as low as 10 Hz have been successfully used. Another promising technique, already used [67] is the creation of nanoparticles that are then rapidly thermally evaporated on a hot surface. This technique produces less fragmentation, because the thermal energy for evaporation is converted to translational energy efficiently, while the vibrational and rotational modes remain cold; thus the molecules have low internal energy, and are less likely to fragment on photoionization. Another technique that has been used in optical laser experiments is to embed the biomolecule in a helium droplet [68]. Although the molecule is evaporated thermally, it is then picked up by an extremely cold liquid helium droplet, after which it is cooled by evaporation, so that its internal energy is extremely low. Although this technique is often used in the laser community, it is in its infancy for synchrotron and FEL studies. The state of the art technique to bring unfragmented nucleotides, proteins or peptides from solution into the gaseous phase is represented by the Electrospray Ionization (ESI) technique [69, 70]. The process consists of: a) liquid injection into a capillary biased at high voltage, from which it exits into a low 16 Page 16 of 23

pressure gas, b) charging of the liquid that explodes into tiny droplets, due to Coulomb repulsion c) drying of the droplets by collision with the gas stream producing solvent evaporation and subsequent fission into progressively smaller droplets due to Coulomb repulsion until the limit of multiply charged single molecules is reached; d) passage through an aperture into a high vacuum section. The ESI source overcomes the difficulties of introducing large molecules prepared in solution into a mass spectrometer, which works in a

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high vacuum environment. Since the commercial development of ESI sources, mass spectrometry has become the most popular tool for the study of very large organic molecules. The coupling of this versatile

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ion source with spectroscopic techniques implies that the mass selected ions generated in the gas phase by an ESI source are collected in an ion trap and then excited with IR and UV laser pulses [71-73]. Despite the

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low density of the target beam, which hampers the achievement of a good signal to noise ratio, with the available photon flux in third generation synchrotron sources, some pioneering works using mass spectrometric techniques have been reported [74-80]. For example Gonzalez-Magana et al. [81] used a

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combination of an ESI source, tandem mass spectrometer and Paul trap to measure partial ion yield NEXAFS spectrum at the carbon edge of protonated leucine enkephalin, a peptide containing 5 amino acid residues. Using similar methods to produce the sample, Milosavljević et al. [77] produced free protonated

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cytochrome ions (consisting of 104 amino acids, and charge state selected), and performed VUV photoionization mass spectrometry. An improvement of the throughput of the sources is needed in order favour the use of ESI sources.

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to make them suitable for electron spectroscopies. The high intensity of the new FEL sources will certainly

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The environment can strongly amplify/reduce the effects of the ionizing radiation and in general affect the physical-chemistry behaviour of the target molecule. Thus sources of dry or hydrated clusters of these

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molecules have been developed. An example is the gas aggregation cluster source [82], used at CIMAP Caen (France) for mass spectrometric studies in ion impact experiments, where the powder of the target sample is resistively heated in an oven and then a He buffer gas carries the vapour of the target molecules into a liquid nitrogen cooled condensation channel, where the molecules aggregate. The cluster distribution produced depends on various parameters [83], and in order to produce hydrated species, the He beam can be seeded with water molecules. Thus, a He/H2O mixture flows into the condensation channel with the biomolecules, and hydrated clusters are produced. The present limit of the technique is the absence of mass selectivity in the target beam, which however may be in part overcome by electron-ion and ion-ion coincidence techniques. Another source is based on a supersonic jet expansion in which the carrier gas is bubbled through water and passed over the sample vapour. This has been developed at the Advanced Light Source and successfully used to investigate proton transfer in dry and microhydrated clusters of nucleobases [84]. An ESI source can be also fruitfully used to generate van der Waals clusters of molecular systems surrounded by a controlled number of water molecules. In this way a solvated environment can be mimicked in the gas phase [85] and its effects on the structure and properties of the biomolecular system 17 Page 17 of 23

investigated. As stated in the Introduction, gas phase studies enable the distinction between intrinsic properties of the molecules and those due to the interaction with the environment. The combination of synchrotron light and more sophisticated sample sources, which allow the preparation of hydrated sample for example, provide direct information about biomolecules in their natural environment, that is, aqueous solution. This

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is a promising area for future development.

Nanoparticles are increasingly used in the biological field for drug delivery due to their functional

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surface, which gives them the ability to bind, adsorb and carry other compounds [86]. Moreover their enhanced permeability and retention and the ease with which they are taken up by cells favours them with

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respect to other carrier systems. Among nanoparticles, noble metal nanoparticles are preferred due to their optical properties, non-toxicity and biocompatibility compared to the other metals. Wang et al. [87] have presented a comprehensive review of synchrotron radiation based techniques for the study of

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nanomaterials at cellular and subcellular interfaces and their transformation in-vitro as well as of the molecular mechanism of the reaction of nanomaterials with biomolecules, but studies of isolated nanoparticles are still rare. At the Chemical Dynamics Beamline of the Advanced Light Source [88,89] angle

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resolved threshold photoelectron spectra and elastic scattering of VUV light have been used to characterize NaCl nanoparticles of 25-350 nm. The experiments have been extended to the soft X-ray region at Soleil

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[90]. Recently Xiong et al. [91] coupled a nanoparticle aerosol source to a femtosecond velocity map imaging photoelectron spectrometer. In this way gas-phase photoelectron spectroscopy techniques have

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been used to study the evanescent exciton wave function in colloidal quantum dots, which typically must be studied in a liquid solvent or while bound to a surface. These results show that synchrotron radiation

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techniques applied to isolated systems can provide additional information about the electronic structure and local chemistry of nanoaggregate and therefore they can find a useful application in the functionalised nanoparticles that are used/proposed for drug delivery. A step forward in understanding the dynamics and energy flow in biomolecules involves the time

resolved study of the processes occurring between the energy absorption and the manifestation, for example, of the damage or de-excitation and ‘repair’. This will lead to the control and proper handling of the process itself. A typical example is represented by the study of the mechanisms that nature has provided to DNA/RNA bases in order to avoid that their strong absorption in the UV region (at about 260 nm) leads to their destruction. These processes occur on ultrafast timescales. For this purpose, excitation sources with the appropriate wavelength and pulse duration and, more important, fast and powerful probe tools are needed. The third harmonic of fast Ti-sapphire lasers can be used as excitation sources and then several optical schemes [92, 93] have been adopted for the probe that ionizes the excited molecules at different delay time with respect to the pump. These works have provided some understanding of the typical time scale of the energy redistribution in nucleobases. However ultrafast pump-probe 18 Page 18 of 23

spectroscopies using optical pulses can suffer from a reduced Franck–Condon overlap upon vibrational relaxation during the evolution of the wave packet on the potential energy surface. This problem can be overcome by the use of inner shell spectroscopies, which work at energies far from the ionization threshold and so are capable of core-ionizing the molecule at any nuclear geometry. Due to their element- and siteselectivity, they allow observation of the nuclear as well as the electron dynamics of the molecule from a

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highly local point of view. The Free Electron Lasers (FEL) now operational in the the soft X-ray region with pulses of the order of a few tens of fs have the possibility to perform these experiments. The first example

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is represented by a study of Thymine performed at the LINAC Coherent Light Source (LCLS) [94]. In that experiment the spectrum of the O KVV Auger electrons has been used to probe the initial elongation of a

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C–O bond after molecular photoexcitation. The elongation manifests itself as a shift of the Auger spectrum to higher kinetic energies. The results of these measurements allowed one to deduce an initial photoexcited state lifetime of 200 fs and to demonstrate that a probe that uses ultrafast Auger spectra is

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able to distinguish between nuclear relaxation and non-Born Oppenheimer Approximation electronic transitions.

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5. Conclusions

In this brief review it has been shown how the tunability of synchrotron radiation combined with

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electron spectroscopies and mass spectrometry has been exploited for the characterization of the

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electronic structure, the conformations, and the dynamic processes that lead to the redistribution of the energy absorbed by molecules of biological interest, such as amino acids, peptides and the building blocks

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of nucleic acids. The advent of more intense and short pulse sources such as FELs will enlarge the number of molecules to be investigated, overcoming the limitation of the low density of the beams produced by novel methods. Further, it will be possible to follow the evolution of the energy flow in the molecule as well as processes like H migration, molecular dynamics (rotation or bending of bonds, etc) which can strongly affect the chemical behaviour of these molecules or their fragments.

Acknowledgments

Work partially supported by the MAE Italia-Serbia Joint project of particular relevance “Nanoscale insights in radiation damage” and the COST Acion XLIC.

19 Page 19 of 23

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