Time‐resolved x‐ray spectroscopy of nucleobases and their thionated analogs

The photoinduced relaxation dynamics of nucleobases and their thionated analogs have been investigated extensively over the past decades motivated by their crucial role in organisms and their application in medical and biochemical research and treatment. Most of these studies focused on the spectroscopy of valence electrons and fragmentation. The advent of ultrashort x‐ray laser sources such as free‐electron lasers, however, opens new opportunities for studying the ultrafast molecular relaxation dynamics utilizing the site‐ and element‐selectivity of x‐rays. In this review, we want to summarize ultrafast experiments on thymine and 2‐thiouracil performed at free‐electron lasers. We performed time‐resolved x‐ray absorption spectroscopy at the oxygen K‐edge after UV excitation of thymine. In addition, we investigated the excited state dynamics of 2‐tUra via x‐ray photoelectron spectroscopy at sulfur. For these methods, we show a strong sensitivity to the electronic state or charge distribution, respectively. We also performed time‐resolved Auger–Meitner spectroscopy, which shows spectral shifts associated with internuclear distances close to the probed site. We discuss the complementary aspects of time‐resolved x‐ray spectroscopy techniques compared to optical and UV spectroscopy for the investigation of ultrafast relaxation processes.


INTRODUCTION
Nucleobases and their thionated analogs play a crucial role in life.As core constituents of the DNA, nucleobases encode the genetic information and participate in different parts in metabolism.Nucleobases exhibit large UV absorption crosssections.In DNA, such UV-induced excitations can lead to the formation of intrastrand dimers between neighboring nucleobase pairs. 1 These UV-induced lesions affect the reproduction of DNA and can eventually lead to mutations and cell death.However, the low quantum efficiency for these processes limits the rate of formation of such dimers.Already in isolated nucleobases, one observes a competing relaxation mechanism: ultrafast relaxation from the photoexcited state back into the ground state provides a protective channel 2,3 by dissipating the photoexcitation as heat.For pyrimidine lesions, time-resolved UV pump-infrared probe studies show the complex interplay that ground-state relaxation and dimerization play in photoprotection. 4pon thionation, that is, the substitution of oxygen atoms with sulfur, the photophysics and photochemistry of nucleobases significantly change.Strong redshifts in the absorption spectrum allow thionucleobases to absorb in the UV-B or even UV-A spectral range.6][7][8] In contrast to their canonical counterparts, they efficiently relax into long-lived triplet states that promote intrastrand crosslinking or photolesions, which can be exploited for photodynamic therapy. 9However, these very same features make these molecules dangerous in their other forms of medication (immunosuppression) due to the abundance of UV-A in terrestrial sunlight.4][15][16] Various experimental techniques have been used to investigate the electronic and nuclear dynamics and have been combined with extensive theoretical calculations to understand relaxation after UV excitation. 14,15Conical intersections (CI) play a key role in the relaxation dynamics of these molecules. 17,18These are regions in the potential energy landscape where the potential energy surfaces of two different electronic states approach each other and intersect.In these regions, the Born-Oppenheimer approximation (BOA) breaks down.In short, the BOA allows the decoupling of electron and nuclear motion in quantum mechanical calculations as it assumes that the much lighter electrons can immediately react to any changes in the molecular geometry.However, as the energy gap between two PES decreases, the time scales of nuclear and electronic motion converge, coupling the two degrees of freedom.This allows the electronic wavepacket to release excess energy from optical excitations by changing molecular geometry and electronic state.Therefore, the shape and gradient of the PESs and which minimum can be reached fastest have a large influence on the relaxation pathway.In general, relaxation via CI often dominates over radiative deactivation channels as fluorescence happens on much slower timescales.
In many instances, the search for the exact relaxation pathways remains a struggle.Experimental techniques that focus on studying only the valence electrons often cannot distinguish between electronic and nuclear degrees of freedom and have difficulties identifying electronic states involved in the relaxation without expensive theoretical calculations.This might lead to the proposal of multiple relaxation pathways that are all compatible with experimental findings, as was the case for thymine. 19,20ith the evolution of x-ray free electron lasers over the past two decades, it became possible to investigate molecular dynamics from a completely different perspective using core-level electrons.FELs not only provide ultrafast, intense light pulses, but the high photon energies of hundreds to thousands of eV allow for probing highly localized core-level electrons inside the molecules.The element and site-selectivity that comes with the use of x-ray pulses allows for a systematical probe of different locations in the sample and provides new information that allows disentangling electronic and nuclear degrees of freedom, giving more direct insight into the relaxation dynamics of UV excited molecules.][23][24][25][26][27][28] When studying the relaxation dynamics of molecules with x-rays, the usage of the gas phase comes with some advantages.First, the experiment can be often directly compared to theory.The simulation of isolated molecules can be accomplished at a higher level in approximation and, hence, a comparison is easier and faster.Second, the low density of gas-phase targets allows for different experimental techniques to be employed on the molecular problems and, hence, offers the possibility of creating a more holistic picture of the problem.Excited-state absorption, which is commonly used in solution-phase, is more time-consuming in gas-phase due to the low density.The detection of charged particles, that is, electrons and ions, gives access to different "mapping" of the molecular dynamics of observables.In contrast to optical transitions among molecular electronic states in time-resolved absorption, the dipole selection rules in photoelectron spectroscopy are less confining due to the availability of ionization continua of different angular momentum. 291][32] In the solution phase, ion detection is prohibitive and electrons can, depending on their energy, only be collected from a rather thin surface layer.In addition, depending on the elemental composition of the solvent, the SXR pulses are strongly absorbed in the solvent.
In this review, we summarize our studies on thymine (Thy) and 2-thiouracil (2-tUra) performed at x-ray freeelectron laser facilities over the past decade.We will start with a brief overview of the proposed relaxation pathways of both molecules based on the valence spectroscopy and simulations performed before.Subsequently, we discuss different methodological approaches in time-resolved xray spectroscopy for gas phase targets.We start with x-ray absorption, which allows us to disentangle the different electronic states involved in the relaxation.Then, we continue with photoelectron spectroscopy which is to deduce the localized charge at the probed atom via the observation of the Excited State Chemical Shift (ESCS).At the end, we discuss what can be learned from the Auger-Meitner decay that follows the core-ionization of UV-excited molecules.

NON-ADIABATIC RELAXATION DYNAMICS OF THYMINE AND 2-THIOURACIL
In this section, we briefly review relaxation pathways for thymine and 2-thiouracil as proposed by different theoretical studies.This is accompanied by a summary of experiments performed in both the gas and solution phases for the respective molecules.However, this summary is limited to experiments that use IR, visible or (V)UV light as the (soft) x-ray spectroscopy will be discussed in later parts of this review.
The relaxation of molecules in the excited states is determined by the shape of their potential energy (hyper-) surfaces (PES) and in particular by the minima, saddle points, CI and potential energy barriers, which can affect the relaxation of the system.During radiationless relaxation, the nuclear wavepacket explores the PES by following the gradient toward lower energy, starting from the Frank-Condon region of the initially excited electronic state.This minimum energy path approach is a standard method to investigate the PES from a static perspective and to get an idea of possible reaction pathways and their time scales. 33The role played by nonadiabatic coupling between states such as CI is often studied with dynamical simulations such as semiclassical surface hopping or multiconfigurational time-dependent Hartree. 34,35n experiments, nucleobases have shown weak fluorescence yields after optical excitation. 36This indicates that non-radiative deactivation mechanisms dominate.Theoretical studies describing possible pathways are summarized in Ref. [16].In short, the excitation with UV light populates a ππ* state in these molecules.nπ* states, which sometimes are lower in energy, show weak oscillator strength for optical excitations due to the lone pair orbital n involved.However, CI between these states and the ground state, often in the vicinity of the respective minima, facilitate non-radiative transitions with femtosecond to picosecond lifetimes.While tautomerism can have an effect on the relaxation dynamics of the molecule, pyrimidine-based nucleobases uracil and thymine, show negligible tautomerism even at high temperatures. 37,38or thymine, a sketch of the proposed relaxation pathway is shown in Figure 1A.A strong UV absorption at ca. 265 nm (4.67 eV) prepares an electronic wavepacket in the ππ* state (i.e., an electron is promoted from the π into the unoccupied π* orbital).From the Franck-Condon region the molecule will start to explore the new potential energy F I G U R E 1 Schemes of the potential energy surfaces of thymine (A) and 2-thiouracil (B).Canonical pyrimidine nucleobases such as thymine show ultrafast relaxation into the ground state after a UV excitation due to conical intersections (CI) among ππ* and nπ* states and the ground state that allow for non-radiative transitions between electronic states.Thionucleobases such as 2-tUra, in contrast, relax efficiently into triplet states because a CI with the ground state is located at a higher energy than the state minimum and a stronger spinorbit coupling introduced by the sulfur facilitates intersystem crossings close to the minima.Schemes adapted from figure 1 in Ref. [39] and figure 4 in Ref. [14].Experiments have shown that thymine relaxes efficiently into the ground state within picosecond timescales without fluorescing, 40 indicating a relaxation via CIs.Over time, different pathways for that relaxation have been proposed.Some of the calculations suggested a direct ππ* to ground state CI. 41,42Others proposed an additional, intermediate, "dark" nπ* state through which the ground state eventually will be repopulated. 20,43,44In addition, the transition time constants were under question too, as some of the calculations predicted a local ππ* minimum which would increase the relaxation into the ground or nπ* state from below 100 fs without barrier to a few picoseconds. 20,41,43xperiments on the photoexcited molecule's electronic states show a variety of time constants ranging from femto-to nano-or even microseconds.An early solution phase experiment performed by Reuther et al. using femtosecond absorption spectroscopy found a 1.2 ps lifetime for the lowest excited state. 45Fluorescence measurements by Gustavsson et al. gave lifetimes of ca.600 fs. 46,47Hare et al. measured the lifetimes of thymine using femtosecond transient absorption and infrared spectroscopy and observed lifetimes of 2.8 ps, 30 ps 40 and 0.56 μs 48 which were attributed to relaxation into the ground state from the 1 ππ*, 1 nπ* or the triplet manifold, respectively.TR-PES experiments in liquids by Buchner et al. yielded lifetimes of 70 and 410 fs which were attributed to different CI between the 1 ππ* and the ground state. 49None of the photoelectron features were attributed to the nπ* state in this study.Timeresolved infrared spectra taken by Manna et al. suggested a triplet formation within 4-6 ps depending on the solvent and a very fast ππ* to ground state relaxation. 50The fraction of molecules that ended up in the triplet state varied between 4% and 16% depending on the solvent as well.In a very recent study, Miura et al. performed EUV-TRPES on aqueous thymine and observed four time constants for the relaxation. 51The ππ* contribution had to be fitted with two lifetimes of 150 and 160 fs.An nπ* contribution was found with a lifetime of 2.5 ps.Also, a long-lived species with a lifetime larger than 20 ps was observed.
For gas phase experiments, comparable values were observed.A study by Kang et al. gave a time constant of 6.4 ps for the relaxation, which was attributed to depopulation of the nπ* state. 52Another long decay with >100 ps was assumed to originate from a small number of molecules ending up in triplet states.6][57] An initial ultrafast relaxation, which ranges between 40 and 500 fs, is followed by a slightly slower relaxation of up to 10 ps.A third very long decay was observed in the experiments but accurate values could not be given.The assignments of the lifetimes followed mostly the suggestion from Ullrich et al. 19,58 : The ππ* state decays within a few ten or hundred femtoseconds and populates the nπ* state.Within a few picoseconds, this nπ* state decays either back into a hot ground state via a conical intersection or undergoes intersystem crossing (ISC) into a (ππ*) triplet state which is supposed to be responsible for the observed nanosecond time constant.
The UV-spectroscopy of photoinduced dynamics of thionated nucleobases differs significantly from their canonical counterparts.The introduction of the sulfur atom lowers the potential energy surfaces in these molecules leading to a significant red-shift in the UV absorption. 14,15urthermore, the increased spin-orbit coupling introduced by the sulfur makes inter-system crossings much more likely.Ultrafast ISC times of a few hundred femtoseconds lead to an increased ISC yield close to unity for most of the thionated nucleobases. 15The triplet lifetimes span from picoseconds to microseconds.
0][61][62][63][64][65] A scheme for a potential energy landscape of 2-thiouracil is shown in Figure 1B.Calculations show that the first broad absorption band around 270 nm (4.6 eV) is dominated by transitions into two close-lying ππ* states with slightly different electron localization and similar oscillator strength. 66,67However, Mai et al. state that excitations of higher lying ππ* states will relax very quickly into the lowest singlet 1 ππ* state so that their internal dynamics do not play a role in subsequent processes. 66Interestingly, Mai et al. predict two minima for the lowest 1 ππ* state: one showing strong pyramidalization at the sulfur site (π S π 2 *), which can also be accessed from higher-lying states, and the other showing puckering and pyramidalization at the C 6 atom (π S π 6 *). 66Both, however, show a CI with the 1 nπ* state close to their respective minima.According to the calculations, the minimum corresponding to the π S π 6 * electronic character might be preferred upon UV excitation. 66,68Conical intersections between 1 ππ* and the ground state have been predicted but are at higher energies compared to their canonical counterparts. 14In the vicinity of the 1 nπ* minimum, the stronger spin-orbit coupling introduced by the sulfur facilitates an ISC into 3 ππ* triplet states.Again, a CI between 1 nπ* and the ground state is predicted but only reachable via an energy barrier.Intersystem crossing between the ground state and lowest 3 ππ* state is predicted above the 3 ππ* minimum trapping possible population in the triplet manifold.
Experiments, focusing again on probing via valence electrons, support the relaxation pathways proposed by Mai et al. 66,68,69 or Cui and Fang, 70 that is, the relaxation from 1 ππ* via 1 nπ* into the triplet manifold.Solutionphase transient absorption spectroscopy studies observe two decay constants. 62,63The first is in the order of a few hundred femtoseconds and is associated with the ISC to the triplet manifold.The response functions in both experiments were not short enough to resolve faster relaxations, and, hence, the 1 ππ* → 1 nπ* relaxation which is predicted to happen within 100 fs could not be resolved.The second time constant observed is in the order of tens of picoseconds to nanoseconds and is attributed to the triplet lifetime.Gas-phase experiments on 2-tUra were conducted using valence photoelectron spectroscopy 55,61,65 and photoions. 59,64All of the experiments show three very similar time constants for their observables.The first relaxation happens within less than 100 fs and is attributed to the depopulation of the 1 ππ*.The second constant is in the order of a few hundred femtoseconds (200-800 fs) and fits the predicted time-scales for the ISC from the 1 nπ* to the triplet manifold.The triplet lifetime is measured to be in the order of 100-200 ps through all the experiments.

PROBING ELECTRON AND NUCLEAR DYNAMICS WITH X-RAYS
In this following section, we briefly discuss the basics of the x-ray spectroscopic methods used, as well as their applications on thymine and thiouracil.X-ray probes provide an advantage over optical probing techniques due to element sensitivity and site specificity.Thus, ultrafast x-ray probing allows for a localized view of the molecular dynamics.
Core-level energies differ significantly between elements.For the most important elements in organic compounds S, C, N and O, the 2p and 1s binding energies are separated by over 100 eV starting from around 160 eV for S 2p, 290 eV for C 1s to 410 eV for N 1s and 540 eV for O 1s.The x-ray-induced transitions are confined to small electronic core wavefunctions around the respective atoms in molecules, lending the scheme high spatial-or sitefidelity.For instance, probing the sulfur 2p core electrons in 2-tUra via x-ray photoemission gives direct sensitivity to local changes in the vicinity of the sulfur site in 2-tUra.In x-ray photoelectron spectroscopy (XPS, see Figure 2A-C), one uses this characteristic binding energy and thus the characteristic kinetic energy E kin of the photoelectron to detect signals originating from the atom of interest.To investigate excited state dynamics, a UV pump x-ray probe scheme can be used.Figure 2A shows an orbital scheme of XPS, with and without a UV excited valence state.The scheme in Figure 2B displays the pump-probe measurement scheme in a multi-electron picture, highlighting the dependence of the binding energy on the nuclear geometry.The UV pulse launches the molecule on the valence-excited PES.Afterwards, the x-ray pulse leads to a core-level cationic state and an outgoing electron with a kinetic energy equal to the difference between the photon energy and the binding energy of the core level.
Site selectivity is even possible when multiple atoms of the same element are present in the molecule.This is due to so-called chemical shifts, that is, eV-scale variations in the core-binding energy (and thus XPS kinetic energy) driven by electronegativity variations in the atom's environment. 71,72The electronegativity of different atoms in the molecule affects the distribution of the valence electrons over the molecule.This, however, impacts the screening of the nuclear charge for core-level electrons and therefore changes their binding energy with respect to the isolated atom.A prime example of this is ethyl-trifluoroacetate (C 4 H 5 F 3 O 2 ), where the four C 1s peaks spread over a 10 eV window. 71,73Similar effects can also be observed in nucleobases and their thionated analogs. 38,74,75In addition to the chemical shifts observed in molecules when compared to isolated atoms, the binding energy of core levels can be affected by excitations in the electronic state.The changes in electronic density caused by valence excitations affect the screening of nuclear charge that is felt by core electrons.We can exploit this change to follow dynamical processes, by monitoring the changes in core level binding energy as molecular relaxation occurs.Figure 2C shows the redistribution of electric charge on the sulfur atom in 2-tUra that follows photoexcitation.The sensitivity of time-resolved XPS to Excited-State Chemical Shifts (ESCS) and how they relate to the electronic states will be discussed later in this review on the example of 2-tUra.
The concepts of element-and site-specificity discussed in the case of XPS also carry on to other x-ray methods, such as to x-ray absorption spectroscopy.Here, a core-level electron is promoted into unoccupied valence orbitals upon absorption of an x-ray photon.As those core-to-valence transitions appear close to the core ionization potential, the method is also called near edge x-ray absorption fine structure (NEXAFS) spectroscopy. 76Experimentally, NEXAFS spectra can be measured either by detecting the transmitted light or the photoproducts, that is, electrons or ions, as a function of photon energy.The scheme for NEXAFS spectroscopy is shown in Figure 2D-F, again in a single-electron orbital picture (D) and multi-electron PES (E).The transition dipole of the excitation depends significantly on the orbital overlap of the involved core and valence orbital.As the core electrons are strongly confined around individual atoms, transitions will only occur into valence orbitals that have significant electron localization at the respective site.Thus, lone-pair orbitals, such as the sulfur n orbital, indicated in Figure 2F, showing a more atomic character, will show a stronger absorption from respective sites than more delocalized valence orbitals such as π orbitals.This can be very helpful for the investigation of relaxation dynamics of molecules where nπ* states play a crucial role.
After the creation of a core hole, the molecule is left in a highly excited state.Weaker-bound electrons will reoccupy the inner-shell vacancy within tens of femtoseconds.The excess energy can be dissipated in two ways: by emitting a photon or by self-ionization of another valence electron (see Figure 2G).The first process is known as xray fluorescence and primarily occurs in elements with high nuclear charge ("heavy atoms") as can be deduced from the Einstein coefficients. 76For lighter atoms such as C, N, O and also S which are the main components of organic molecules, the emission of valence electrons via the Auger-Meitner (AM) effect 77,78 is favored.The energy of the emitted electrons only depends on the binding energies of the involved orbitals that is, the core level and the  valence orbitals.Thus, AM spectroscopy is insensitive to the photon energy and its experimental fluctuations, giving an advantage when compared to the above-mentioned techniques.Attributing spectral features to the plethora of AM transitions, however, requires comparably elaborate theoretical calculations.The decay of the core hole via AM-transitions involves valence electrons with a high localization at the core-hole site.Thus, AM spectroscopy is sensitive to structural changes in the vicinity of the atom.Since a major part of the total energy of the (di-)cationic final states is stored in the Coulomb repulsion between the positively charged neighboring atoms, the bond distance affects the resulting AM electron kinetic energy.This sensitivity, however, only allows for qualitative statements on geometric changes.For more quantitative results on the nuclear configuration, time-resolved diffraction experiments are better suited to study the geometry (changes).[86][87]

Experimental setup
The experiments that will be discussed in the following sections were conducted at different free-electron laser facilities, namely the linac coherent light source (LCLS) at SLAC National Accelerator Laboratory, USA, 22 and the FLASH free electron laser at DESY in Hamburg, Germany. 21,88,89Free-electron lasers are powerful tools for the measurements as they provide short (femtosecond) and intense light pulses in a broad spectral region reaching from the THz to hard x-ray domain.
The experimental setup for time-resolved XPS, NEXAFS and AM are identical and determined by the fact that the sample consists of isolated molecules.In the following, we will briefly describe the experimental scheme.Details of the apparatus at LCLS can be found in Refs.[39, 90] and for FLASH in Ref. [91].A sketch is shown in Figure 3.The molecules that are solid at room temperature are brought into gas phase via an in-vacuum oven system described in Ref. [39].With this system, the molecules can be heated up to ca. 150°C and the vapor is directed to the interaction region with a small capillary at the end of the oven.
The molecules are excited with a UV pump pulse with a wavelength of around 270 nm and sub-100 fs pulse duration.The pulse energy is usually chosen in a way such that only a fraction of the molecules are excited to avoid nonlinear effects.An x-ray pulse provided by the FEL is tuned to core-excite or ionize the respective atoms probes the molecule.Photo-and Auger-Meitner electrons are collected and their kinetic energy is determined using a magnetic bottle electron time-of-flight spectrometer (MBES).The MBES allows for a high collection efficiency of almost 4π, due to its inhomogeneous and guiding magnetic field. 92This large collection efficiency is favorable for dilute gas phase spectroscopy.

Time-resolved NEXAFS spectroscopy
We now present time-resolved NEXAFS spectra of thymine at the oxygen edge.The experiment delivers information that is complementary to the time resolved absorption and valence photoelectron spectroscopy.The static x-ray absorption spectra of isolated thymine at the oxygen edge observed in an experiment at LCLS (Figure 4A, black line) 93 agree with synchrotron measurements. 74Two absorption peaks are observable at 531.5 and 532.5 eV photon energy which can be attributed to transitions from the oxygen 1s into linear combinations of π* orbitals which conserve the high localization of electrons at the respective oxygen atom.
We now concentrate on the effect of molecular UV excitation on the NEXAFS spectra. 93The result for 2 ps delay between UV pump and x-ray probe pulse is shown  The transition from a core-orbital to a lone pair orbital at the same atom is very strong, stronger than corevalence transitions to delocalized orbitals.Intuitively one would expect that the oxygen 1s-n transition dominates the UV-induced NEXAFS feature, and this was also confirmed by a transition-strength calculation in Ref. [93].This strong atomic absorption feature has also been used in a liquid jet experiment of water radiolysis. 94e two-dimensional false color plot in Figure 4B shows the time-resolved bleach at the 1sπ* resonance (531 eV, blue) and rise of the 1s-n resonance (526.5 eV, red).This reveals the dynamics of the ππ*-nπ* relaxation.The bleach at zero delay of pump and probe indicates the UV induced ππ* excitation.The 1s-n absorption signal, reflecting the nπ* population, occurs with a delay of ca.60 fs.We therefore attribute a time constant of 60 fs to the ππ*-nπ* relaxation in agreement with the first attributions 19 but in strong contrast to the picosecond constant predicted later. 20,43,95The 60 fs time constant for the ππ*nπ* relaxation demonstrates a very direct path for access to an efficient internal conversion.
Transient x-ray absorption spectroscopy is an important technique in the hard x-ray domain and it is mainly used in the context of metal-containing molecules in liquid environments. 96,97In the gas phase and the soft x-ray domain, several ultrafast experiments using inherently broadband high harmonic generation sources have been performed.They investigate, for instance, strong field ionization dynamics and UV-excited ring-opening reactions. 98

Time-resolved ray photoelectron spectroscopy
The electronic relaxation molecules through different electronic states after excitation is often accompanied by significant movement of valence charge around the molecule.This relocation of electric charge affects the screening of nuclear charge for strongly localized electrons and, hence, will impact their binding energy.In the case of 2-thiouracil, the two highest occupied molecular orbitals, the π and the n orbital, show significant localization at the sulfur atom.The unoccupied π* orbital-which is the electron's destination for the first optically excitable transition in the molecule-shows, in contrast, a strong delocalization over the pyrimidine ring.An excitation from π to π* will, thus, shift electron charge from the sulfur to the ring.This is shown in the charge difference maps in Figure 5A.The red color indicates a more positive charge with respect to the molecular ground state and the blue more negative charge.The sulfur indeed loses valence charge while the neighboring carbon atom seems to receive most of it.With the decrease of screening at the sulfur, it can be expected that core-localized electrons at the sulfur, that is, 1s, 2s, and 2p, will increase their binding energy due to a higher Coulomb force.Vice versa, the C 1s electron would be expected to reduce its binding energy due to an increased screening.
We have measured the time-resolved photoelectron spectra at the sulfur site of 2-tUra at FLASH free electron laser. 102Figure 5B shows the static (blue) and pre-excited (orange) spectrum for the S 2p photoelectrons.Due to the relatively large bandwidth of the FEL, the spin orbit splitting of the photoline cannot be resolved.Synchrotron data (shown in black) shows the fine structure in the electron spectrum.The photoline is accompanied by a small satellite feature at lower kinetic energies originating from shake-up processes during the ionization.The UV excitation of the molecule alters the shape of the spectrum.The main photoline bleaches and new signal rises at lower kinetic energies.The shift of the excited state signal amounts to ca. −3 eV.This indicates an increase in the binding energy for the S 2p electrons, which agrees with the above made prediction.
The observed shift is an immediate response after the excitation and lasts for at least 100 ps, which agrees with previous results on the lifetime of the triplet states.Figure 5C shows the excited state signal for debetween pump and probe pulse as a false color map.Following the contour lines, a change of the shape of the feature can be observed within the first picosecond of the relaxation that follows an oscillatory behavior.The band broadens towards lower kinetic energies and contracts again over a course of ca.250 fs.Such changes in kinetic energy can indicate changes in the electronic character of the excited state during the relaxation.Here, it can be an indication of coherent charge flow between ππ* and nπ* states as a hole in the lone-pair orbital would decrease the screening at the sulfur further.Previous calculations by Mai et al. also show oscillatory behavior in the population of the nπ* states, 68,69 which match the experimental oscillation strengthening the assumption of coherent charge flow due to changes in the electronic state.
Coupled-cluster calculations, which we performed for the study, confirm the strong correlation between local valence charge, core-level binding energy and electronic states.Both singlet and triplet nπ* states show the highest partial charge at the sulfur and also the highest shift in binding energy while ππ* states give rise to less but still significant redshifts.The calculations also suggest that geometrical changes during the relaxation play little to no role.
Studies on time-resolved XPS on small molecules are still rather limited but other recent experiments show the possibilities the method offers.In 2018, Leitner et al. 103 and Brauße et al. 104 published the first studies where ultrashort x-ray pulses from FLASH were used to investigate dissociation dynamics of small molecules in the gas phase.Brauße et al. studied the photodissociation of CH3I using velocity map imaging of electrons and ions. 104They were able to identify a chemical shift in the electron spectra that corresponded to the formation of atomic iodine with a rise time of ca.20 fs coinciding with the temporal resolution of their experiment.Leitner et al. studied the photodissociation of iron carbonyls using a magnetic-bottle timeof-flight spectrometer. 103The chemical shifts observed in Fe(CO) 3 , Fe(CO) 4 and Fe(CO) 5 gave insight into the bond changes between the different complexes.
More recent studies have further pushed the capabilities of TRXPS.Allum et al. studied the photodissociation of 1-iodo-2-methylbutane also at FLASH. 105In the experiment, the group exploited the dual-sided velocity map imaging setup of the CAMP endstation at FLASH1 to test a electron-ion partial covariance imaging scheme for photoelectron spectroscopy.Chemical shifts observed in the I 4d photoline could again be attributed to the formation of atomic iodine.Faccialà et al. used circularly polarized x-ray pulses from FERMI to investigate the photoexcited Rydberg states of fenchone by a combination of TRXPS and time-resolved photoelectron circular dichroism (TR-PECD). 106The chemical shift observed in the x-ray photoelectron spectra could be attributed to charge relocation as the highest occupied molecular, which is depopulated upon excitation, is a lone pair orbital localized at the C 1 , C 2 and C 3 atoms.The combination with the PECD, however, appears challenging due to the low signal-tonoise ratio during the experiment.Nonetheless, the UVinduced chemical shift isolated the C 2 /C 3 contribution in the PECD whose dynamics could thus be studied and rationalized.Gabalski et al. recently revisited the photodissociation dynamics of CS 2 using TRXPS at FLASH. 107hey show that the experimentally observed chemical shift upon UV excitation can be attributed to the formation of the photofragments CS and S as their binding energy drastically differs from CS 2 .In addition, they show that TRXPS is also sensitive to the vibronic state of the fragments.Though most of the studies mentioned here face different problems such as limited signal-to-noise or a lack of energy or temporal resolution, they show the capabilities of TRXPS for studying ultrafast relaxation processes and with future improvements of FELs these limitations might be overcome.

Time-resolved Auger-Meitner spectroscopy
We have investigated both Thy and 2-tUra by means of AM spectroscopy. 39,108,109The measurements on Thy were performed at LCLS, those for 2-tUra again at FLASH2.Some results for the time-resolved non-resonant AM spectroscopy of Thy from Ref. [39] are shown in Figure 6.To trigger the AM decay, the O 1s electrons were ionized.The ground-state spectrum is shown in Figure 6A.Two broad bands can be observed between 470 and 520 eV kinetic energy.Upon UV excitation the FEL measurements show a shift of the higher energetic band (see B). Initially, the Auger-Meitner spectrum shifts towards higher energies but it decays fast in the order of 100 fs and a shift towards lower energies is observed.The quick decay of the first feature already suggests that there might not be a significant energy barrier in the ππ* state trapping population.
The initial shift towards higher energies can be explained based on nuclear relaxation.The created dicationic state is highly repulsive due to unscreened nuclear charge around the C-O bond.A higher Coulomb repulsion, however, leads to a bond elongation which in consequence leads to a lower energy of the dicationic final states.The spectrum shifts towards higher kinetic energies.The quick relaxation of these features and the shift towards lower energies with respect to the ground state, however, contrast a further nuclear relaxation and suggest an electronic relaxation.In combination with dynamics calculations, the results again an ultrafast relaxation channel singlet nπ* state.
Similar experiments were performed at FLASH with 2-tUra. 108Here, the sulfur 2p edge was probed and the Auger band was recorded.Again, a fast shift towards higher kinetic energies can be observed upon UV excitation.This shift can be also attributed to a nuclear relaxation.The reduction of nuclear charge screening upon ionization of the S 2p electrons favors a C-S bond elongation to minimize the energy of the cationic states.An elongation of the C-S bond upon excitation was predicted by previous theoretical studies. 66,68Further dynamics of the Auger band, however, cannot be explained by a pure nuclear relaxation and must be the results of electronic relaxation.Simulating Auger-Meitner spectra in detail is relatively elaborate, and this is the reason that very little studies use this as an observable.For low-lying dicationic states, however, this restriction is somewhat eased.
Resonant AM decay can be used to derive the x-ray absorption spectra of molecules as resulting Auger electrons lead to an enhancement in the electron yield and, thus, can indicate resonant excitation channels.The absorption spectra in 93 were derived that way.The underlying resonant AM decays were studied in Ref. and two interfering de-excitation channels are observed.One of these de-activation channels is the population of a ππ* triplet state, which has also been proposed in a previous VUV study. 57

DEVELOPMENT OF THE FIELD
Several developments are currently occurring using ultrafast x-rays as probe methods.First, new, higher repetition rate FEL sources are available with the European XFEL, at an effective repetition rate of 27 kHz, and LCLS II, at a projected repetition rate of 1 MHz.The high repetition rate allows for using charge particle coincidence methods, such as COLTRIMS reaction microscopy 110,111 for timeresolved molecular spectroscopy, and first examples from these sources on molecules in the ground state show the power of Coulomb explosion imaging for retrieving the molecular geometry. 30In addition, the signal-to-noise in any of the spectroscopic schemes presented above will increase, allowing for investigation of dilute samples and also of samples in a liquid phase environment, where an additional background from absorption of the jet poses difficulties at some x-ray edges at current repetition rates.
A further promising development in the FEL community is the scaling of external seeding to higher repetition rates.Currently, all free-electron lasers on self-amplified spontaneous emission (SASE), meaning they deliver noisy spectral and temporal structure that changes with every shot.The FERMI free electron laser 23 presents a famous exception, as it is externally seeded by a UV laser and operates at harmonics of this radiation.This results in high longitudinal coherence and excellent spectral stability.Scaling the seeding scheme from around 100 Hz at FERMI to higher repetition rates will allow to explore many more methodologies, such as resonant inelastic x-ray scattering, 112 ion trap spectroscopy and again coincidence methods.At the FLASH free-electron laser, seeded operation up to 5 kHz effective repetition rate is planned. 113The increased spectral control with more coherent x-ray laser sources will also result in the realization of nonlinear probe schemes [114][115][116] building experimental success with lasing and Raman emission with SASE sources. 117,118ith regard to the development of sample sources, different techniques have been developed in the past to bring large molecules into the gas-phase, for example matrix-assisted laser desorption/ionization 119 or electrospray ionization. 120They find applications so far mostly limited to mass spectrometry.However, there is ongoing work in improving those techniques with respect to sample density, stability and their application in electron or coincidence measurements. 121,122Laser-induced acoustic desorption was already used in femtosecond spectroscopy with laboratory laser sources 123,124 and could be an option at free-electron lasers.[127]

CONCLUSIONS
The advent of short-wavelength FELs in the last decade started the investigation of light-induced, ultrafast molecular dynamics using femtosecond x-ray pulses.Utilizing the element-and site-selectivity of x-rays allows to disentangle electronic and nuclear degrees of freedom during the relaxation and test proposed relaxation pathways more directly.In this review we have discussed the applications of different x-ray spectroscopy methods for investigating the relaxation dynamics of molecules based on our research on the well-studied nucleobase thymine and the thionucleobase 2-thiouracil.
We showed that the ambiguity in the proposed relaxation pathway for the molecule thymine could be solved with the help of x-ray absorption spectroscopy. 93By directly accessing resonant transitions between the oxygen 1s core and the half empty n-valence orbitals, the optically dark nπ* could be observed as a UV-induced feature in the absorption spectrum.In combination with an observed 60 fs delay in its appearance, this technique allowed to unambiguously point towards a sub-100 fs internal conversion from the ππ* into the nπ* state without delays by a previously suggested energy barrier.
We studied the UV-induced excited-state chemical shifts in the x-ray photoelectron spectra of 2-thiouracil.We discovered the connection between shift and valence charge flow over the molecule upon UV excitation. 102This agrees with the classical model of chemical shifts, where the binding energy of core-level electrons is strongly dependent on the local valence electronic charge.We succeed in observing the electronic changes due to internal conversion and ISC that lead to a redistribution of charge over the molecule.Probing the binding energies of core electrons at different sites and elements inside the molecules will allow us to track charge movement completely and will thus create an electronic molecular movie.
Time-resolved Auger-Meitner spectroscopy shows sensitivity to geometrical changes in the vicinity of the core-hole.Both thymine and 2-thiouracil show an initial shift in the Auger-Meitner electron energy towards higher kinetic energies at the O 1s and S 2p edges, respectively. 39,108From electrostatic considerations, this can be attributed to an initial bond elongation between carbon and the respective heteroatom.However, further relaxation dynamics cannot be attributed to geometrical changes, more expensive theoretical models are needed to capture the electronic relaxation that drives AM spectral shifts after the initial bond elongation.In the future, time-resolved Coulomb-explosion imaging and ultrafast x-ray or electron diffraction will allow us to follow the nuclear geometry changes with much higher level of detail.Together with the high-fidelity data on electronic dynamics, this will allow for the reconstruction of the complete electronic and nuclear geometry changes in molecular excited-state dynamics.
In the future, the ongoing development of FELs in terms of repetition rate and seeding will enable a wide range of spectroscopic techniques for molecular samples to study both nuclear and electronic degrees of freedom in a time-resolved manner.Ongoing development on sources for more complex samples will also allow for the investigation of much larger biological systems than presented in this review.

F I G U R E 2
Probing schemes for different x-ray spectroscopic methods addressed in this review and pictograms on how they relate to the molecular problem.The methods covered are x-ray photoelectron spectroscopy (XPS, A-C), near edge x-ray absorption fine structure (NEXAFS, D-F) and Auger-Meitner spectroscopy (AMS, G-I).In the first column (A, D, G), electron balls schemes are shown depicting the x-ray probing mechanism for ground and excited state molecules.The middle column (B, E, H) shows sketches of potential energy surfaces.The right column (C, F, I) shows the molecules and related observables.
Sketch of the experimental setup.A UV-pump x-ray probe scheme was used in all experiments.Photo-and Auger-Meitner electrons were collected using a magnetic bottle time-of-flight spectrometer.
green line).The main observation is the appearance of a new feature at around 526.5 eV.In addition, a very subtle bleach of the original 1sπ* band can be observed around 531.5 eV, which is due to a depopulation of the ground state.However, it is almost entirely compensated by a redshift of the K edge ionization feature in the excited state93 and, hence, only barely visible in Figure4A.The new appearing feature, which is located one UV photon quantum below the main resonance, represents a new absorption channel due to the UV-induced rearrangement of valence electrons.As the UV induces a ππ* transition, a new 1sπ absorption channel opens.In addition, after the ππ*-nπ* relaxation, which is the matter of this investigation, a 1s-n transition will show up in the absorption spectrum.

- 101 F
I G U R E 4 (A) Ground and excited-state NEXAFS spectrum of thymine.The red bump indicates the appearance of a new strong feature in the UV excited spectrum.(B) Delay-dependent false-color map of NEXAFS difference spectra.(C) Integrated regions of interest in the false-color map.A slight shift of the rising edge can be observed between bleach and gain features.This figure was taken from figure 2 in Ref. [93] without changes and reused under a Creative Commons Attribution 4.0 International (CC BY 4.0) License.

F
I G U R E 5 (A) Computed charge difference maps for 1 ππ* and 1 nπ* states.Red indicates more positive charge compared to ground state, blue indicates more negative charge.(B) Photoelectron spectra of 2-tUra for the ground ("UV-off") and excited state ("UV-on") at 500 fs delay as well the difference spectrum.A static spectrum measured at the PLEIADES beamline of the synchrotron SOLEIL (gray) is included for comparison.(C) Delay-dependent false-color map of the positive lobe of the difference spectrum.

F
I G U R E 6 (A) Static Auger-Meitner spectra of thymine.Synchrotron (green) and FEL (yellow) measurements of the ground-state spectrum are shown together with ADC (blue) and CK-CIS calculations (black).The stick spectra (red and cyan) come from the CK-CIS calculations and indicate transitions from the respective oxygen atoms which produce similar Auger-Meitner spectra to those shown.(B) The top panel shows the delay-dependent false-color map of the difference between ground-state and UV-excited AM spectra of thymine.The bottom panel shows two lineouts from the false-color map, which are indicated by the arrows in the plot above.Material taken from figures 2 and 3 in Ref. [39] without changes.