Deciphering the photochemical mechanisms describing the UV-induced processes occurring in solvated guanine monophosphate

The photophysics and photochemistry of water-solvated guanine monophosphate (GMP) are here characterized by means of a multireference quantum-chemical/molecular mechanics theoretical approach (CASPT2//CASSCF/AMBER) in order to elucidate the main photo-processes occurring upon UV-light irradiation. The effect of the solvent and of the phosphate group on the energetics and structural features of this system are evaluated for the first time employing high-level ab initio methods and thoroughly compared to those in vacuo previously reported in the literature and to the experimental evidence to assess to which extent they influence the photoinduced mechanisms. Solvated electronic excitation energies of solvated GMP at the Franck-Condon (FC) region show a red shift for the ππ* La and Lb states, whereas the energy of the oxygen lone-pair nπ* state is blue-shifted. The main photoinduced decay route is promoted through a ring-puckering motion along the bright lowest-lying La state toward a conical intersection (CI) with the ground state, involving a very shallow stationary point along the minimum energy pathway in contrast to the barrierless profile found in gas-phase, the point being placed at the end of the minimum energy path (MEP) thus endorsing its ultrafast deactivation in accordance with time-resolved transient and photoelectron spectroscopy experiments. The role of the nπ* state in the solvated system is severely diminished as the crossings with the initially populated La state and also with the Lb state are placed too high energetically to partake prominently in the deactivation photo-process. The proposed mechanism present in solvated and in vacuo DNA/RNA chromophores validates the intrinsic photostability mechanism through CI-mediated non-radiative processes accompanying the bright excited-state population toward the ground state and subsequent relaxation back to the FC region.


Introduction
The genomic material and the mechanisms that process the disposal of the excess energy attained upon UV-light irradiation are of paramount importance from both biomedical and biotechnological standpoints. The initially populated excited states and their fate along the distinct deactivation routes present in the DNA/RNA double helix chains relate simultaneously to the intrinsic photostability of the genomic material (Callis, 1983;Crespo-Hernandez et al., 2004;Kohler, 2010), as well as to the damaging photo-reactions that ultimately yield mutations and single-and double-strand breaks that have been associated to increasingly featured diseases like skin cancer (Cadet et al., 2012;Noonan et al., 2012;Giussani et al., 2013b;Brash, 2015). Besides the biological relevance, the intricate photophysical properties of nucleobases can be also exploited to design optical photo-responsive nanomachines among a long list of prospective applications (Kamiya and Asanuma, 2014), yet it is their biological relevance that focuses our attention.
From a bio-physical and chemical point of view, fundamental knowledge on the deactivation processes, including the associated underlying molecular motions, are essential to rationalize the intrinsic photostability of the genomic material and to characterize the aforementioned routes leading to deleterious photochemical reactions Cadet et al., 2012). These different pathways increase in number and complexity as the size of the DNA/RNA single-or double-stranded chain enlarges, as the different de-excitation routes interact with one another yielding a ramification of complex photoinduced pathways that become extremely hard to discern, disentangle, and comprehend. This increasing degree of complexity arises partially due to the intermolecular interactions occurring from inter-and intra-strand conformations between the nucleobases compressing the DNA/RNA chains, related to their hydrogen-bonding motifs and π-stacking interactions, respectively. Extensive experimental work has been carried out recently in order to identify and separate the different contributions arising from these interactions, providing indicative fingerprints in the appearance of a long-lived spectroscopic signal originating from a π-stacking conformation within intra-strand sequences (Takaya et al., 2008;Su et al., 2012;Vayá et al., 2012;Chen et al., 2013Plasser et al., 2015) and to an ultrafast proton/hydrogen transfer stemming from a charge-transfer event in the hydrogen-bonding motifs responsible for the inter-strand interactions Temps, 2007, 2008;Saurí et al., 2012). Given the abstruse nature of the photo-processes under study and their intrinsic difficulty, new development in methods and techniques are expected to play a crucial role on disentangling all the different deactivation channels (West et al., 2011Krause et al., 2012;Fingerhut et al., 2013Fingerhut et al., , 2014Rivalta et al., 2014;Nenov et al., 2014a,b), even though the photophysical and photochemical studies on single nucleobases remain at the cornerstone of the field.
A proper characterization of the monomeric chromophores is therefore essential to understand the photoinduced events occurring in DNA/RNA from a bottom-up approach that can yield definitive answers regarding their role in photostability and photo-damage (Serrano-Andrés and Merchán, 2009). This avenue of research embodies the main efforts carried out by the research community over the last two decades (Kleinermanns et al., 2013;Giussani et al., 2013a;Barbatti et al., 2014;Mai et al., 2014), and even though plenty of advances have been made there is still no definitive consensus regarding the main deactivation routes present in the nucleobases (Crespo-Hernandez et al., 2004;Hudock et al., 2007;Barbatti et al., 2010;McFarland et al., 2014). Several decay paths have been identified both in vacuo (Giussani et al., 2013a) and in solution (Improta and Barone, 2014) and assigned to a number of processes, ranging from a barrierless deactivation through a ring-puckering motion of the bright ππ * state (Merchán and Serrano-Andrés, 2003;Gustavsson et al., 2006;Merchán et al., 2006;Serrano-Andrés et al., 2006Conti et al., 2009;Improta and Barone, 2014) to a long-lived signal arising due to a possible crossing with a dark nπ * state (Hare et al., 2007), as well as an even longerlived pathway widely attributed to the role of triplet states, specially prominent in non-canonical nucleobases with heavy-atom substitutions (Merchán et al., 2005;González-Luque et al., 2010;Martinez-Fernandez et al., 2012Pollum et al., 2014).
In this study we turn out attention to water-solvated guanine monophosphate (GMP), one of the canonical DNA/RNA nucleobases. GMP has also been recently proposed to be an interesting compound for nanotechnological applications due to its outstanding capacity for self-assembly (Gupta et al., 2014), specially remarkable in its quadruplex form, which has even been located in DNA/RNA chains (Lam et al., 2013;Murat and Balasubramanian, 2014). Whereas, guanine in vacuo has been extensively studied at a high-level multireference ab initio level (Serrano-Andrés et al., 2008;Barbatti et al., 2010Barbatti et al., , 2011 as well as experimentally (Miannay et al., 2010;Chatterley et al., 2014), a lesser degree of scrutiny, mainly at a density functional theory level (Karunakaran et al., 2009;Parac et al., 2010;Santoro et al., 2010;Improta, 2014;Improta and Barone, 2014), has been considered on the effect of the phosphate group and the the solvent on the photoinduced processes occurring in this compound (Crespo-Hernandez et al., 2004). In the present work we propose a theoretical assessment of the deactivation routes embodying the main photophysical and photochemical features of GMP by employing high-level ab initio multireference perturbation theory methods coupled with a quantum-mechanical/molecular mechanics (QM/MM) approach, in order to ascertain the role of the environmental perturbations in these type of systems as they remain relatively unknown (Conti et al., 2010;Nachtigallová et al., 2010;Barbatti, 2014). Further knowledge on the environmental effects affecting the photo-processes occurring in the DNA/RNA chromophores upon UV-light irradiation will provide essential information that can be properly translated to water-solvated DNA/RNA systems such as those found in cells.

Computational Details
Sections MM Dynamics and Sampling and QM/MM Calculations describe the computational strategies and methodological details considered in the computations of the MM sampling dynamics and electronic structure QM/MM calculations carried out.

MM Dynamics and Sampling
MM simulations were performed for GMP in water to obtain a representative starting geometry to be employed for all subsequent computations and analyses. The MM dynamics calculations were carried out using the Amber-11 suite of programs making use of the Parm99 force field (Case et al., 2005(Case et al., , 2011. Initially, a cubic solvent box comprising 700 water molecules of explicit TIP3P (Jorgensen et al., 1983) with one Na + counterion were considered. The entire system was then heated from 0 to 300 K for 1 ns at constant volume and constant pressure (1 atm), and then finally performing a production run for 100 ns recording the snapshots every 200 fs. To select the initial geometry we performed a cluster analysis based on a Root Mean Square (RMS) coordinate deviation analysis on the guanine moiety over all snapshots recorded along the MM dynamics run within a 2.0 Å difference using the MMTSB toolbox. We obtained three different clusters, denoted a, b, and c in Figure 1, with populations relating to the 93, 6, and 1% of the total number of structures obtained along the dynamics run, respectively. The selected initial structure was therefore chosen as the snapshot with the closest geometrical parameters to the centroid of the average structures obtained in cluster A, being the most representative.

QM/MM Calculations
QM/MM calculations were performed using the COBRAMM interface developed in our group (Altoé et al., 2007a,b). The cut between the QM and MM regions has been done so that it includes all guanine atoms in the QM region, placing the link atom between the N9 of guanine and the carbon of the sugar ring directly attached to the nucleobase (see Figure 2). The choice of the cut between the QM and MM regions is justified in terms of charge redistribution on the nucleobase and its π-system in order to better reflect the covalent link between guanine and the monophosphate group. A three-layer approach (high, medium, and low) was used throughout (Altoé et al., 2007a): guanine was included in the QM region (high layer, ball, and stick representation in Figure 2), whereas the medium layer comprises the movable MM atoms within a 10 Å radius surrounding the GMP moiety, the remainder of the MM system being kept frozen during all optimization procedures in the low layer. Equilibrium geometries and photoreaction paths (Garavelli, 2006) were determined by using fully unconstrained optimizations and minimum energy path (MEP) computations on the relevant potential energy hypersurfaces by employing the intrinsic reaction coordinate (IRC) and optimization algorithms as implemented in the Gaussian 09 program package (Frisch et al., 2009) interfaced with COBRAMM. CI optimizations were performed with the gradient projection algorithm of Bearpark et al. (1994) as implemented in COBRAMM at the QM/MM level. Further details can be found in Conti et al. (2015).
Energies and gradients in the QM region were computed making use of the complete active space self-consistent field (CASSCF) and complete active space second-order perturbation theory (CASPT2) methods (Andersson et al., 1992;Roca-Sanjuán et al., 2012), as implemented in the MOLPRO-2010 (Werner et al., 2012a,b) and MOLCAS-7 (Aquilante et al., 2010(Aquilante et al., , 2013 suite of programs, respectively. All gradient and non-adiabatic coupling calculations have been performed with the CASSCF implementation of the MOLPRO-2010 code. Subsequent CASPT2 calculations on the key structures obtained along the optimizations and MEPs have been carried out as implemented in the MOLCAS package in order to correct the energy values due to the lack of dynamic correlation present in the CASSCF method. This procedure is referred to as the CASSCF/CASPT2 protocol, and has been successfully employed over the years to tackle a variety of photoinduced phenomena from a theoretical standpoint (Garavelli, 2006;González-Ramírez et al., 2012;Segarra-Martí et al., 2012;Segarra-Martí and Coto, 2014). The active space selected comprised the full π space with the exception of the lowest occupied π orbital plus the three lonepair orbitals to provide a proper description of the nπ * states, thus making 18 electrons in 13 orbitals. The removal of the lowest occupied π orbital is carried out due to its occupation number being very close to two and given that its removal does not affect the energy values obtained while speeding up the computations. An imaginary level shift of 0.2 a.u. was employed in the perturbation step to avoid intruder states. Two different basis sets were employed throughout: 6-31G * was used in order to map the hypersurfaces at the CASSCF level whereas atomic natural orbital (ANO) type (Pierloot et al., 1995) basis set with the large (ANO- (ANO-L 321/21 hereafter) was employed in order to refine the single-point CASPT2 energies.

Results and Discussion
The results are divided in three different sections: First, the main geometrical parameters of the ground-state Franck-Condon minimum of 9H-guanine both in vacuo and in solvated GMP are presented, together with its corresponding vertical spectra, drawing some conclusions on the influence the solvent and phosphate group have on the relative position of the excited states. Next, FIGURE 2 | GMP and the three different QM/MM partitions defined in the present study. The high layer treating the guanine moiety at the QM level (ball and stick representation), the medium layer encompassing the movable MM water molecules and the phosphate group enclosed within a 10 Å radius distance from GMP, and the low layer containing the remainder of the MM region that is kept frozen throughout the calculations (see text).
the different minima and conical intersections describing the photo-process are presented. Finally, a rationale of the photoinduced mechanisms in GMP is drawn in conjunction with previous results and experimental data acquired from the literature, yielding concluding notes on the photophysical and photochemical decay channels featuring in GMP and leading to its intrinsic photostability.

UV Absorption at the Franck-Condon Region
The optimized FC structure of GMP in solution (Figure 3) shows a strong resemblance with the in vacuo 9H-Guanine CASSCF/6-31G * structure previously reported in the literature (Serrano-Andrés et al., 2008). Table 1 presents the main geometrical parameters of both structures. As can be seen, bond lengths and dihedral angles are analogous giving rise to a planar structure. Small solvation effects are noticed in the GS structure mainly due to its relatively small dipole moment, providing slight shortenings in the C6-N1 bond distance in the presence of the solvent. A very shallow stationary point (L a ) sp , not present in the gas phase, has been located at the end of the L a MEP featuring a pronounced elongation of the C2-N3 and a shortening of the N3-C4 distances, similar to those featured by the conical intersections between the L a and GS states both in vacuo and in solution. The two CIs located between the L a and GS states do show pronounced differences due to the solvent, yielding large elongations in the C2-N3 bond compared to its in vacuo counterparts. This is mainly due to the large dipole moment featured by the L a state, which makes it a more influenced state upon solvation. A similar behavior can be seen in the CIs between the L a and L b states, prompting large deviations in the C4-C5, C5-C6 and specially pronounced in the C6-O distance, where water-solvated GMP suffers an elongation of 0.15 Å. The n O π * state minimum does not show significant differences highlighting the scarce role polar solvation has on these types of states. The CIs between n O π * and the polar ππ * L a and L b states are profoundly influenced by the solvent comparing them to their in vacuo counterparts, featuring large differences in the C4-C5 bond distance. These differences are mainly attributed to the polar character of the ππ * L a /L b states than to the effects on the n O π * state. Overall, FIGURE 3 | Geometries and main geometrical parameters of the Franck-Condon structure and excited-state stationary points characterized in GMP for its lowest-lying excited states computed at the CASSCF/6-31G* level of theory.
it can be concluded that solvation has an important effect on the polar ππ * L a /L b states, while being negligible for the n O π * state. These states feature excitations that are prominently placed on the six-member ring of guanine featuring noticeable changes in the structure upon solvation, whereas the five-member ring remains relatively unchanged. A possible cause for this effect could lie on the presence of the phosphate group, which is tied to the five-ring member and could be shielding that molecular moiety from the surrounding water molecules thus mitigating its exposure as compared to the six-member ring and justifying why the latter suffers such pronounced changes upon solvation. Table 2 contains the vertical excitation energies at the CASPT2 level computed in the present study, together with several other computations and experimental values reported in the literature. We have used Platt's nomenclature (Platt, 1949), where L a represents the ππ * excited state characterized by the largest contribution of the configuration HOMO (H) → LUMO (L) to the CASSCF wave function, whereas L b depicts the ππ * excited state with a predominance of H → L+1 and H-1 → L configurations. By inspection of Table 2, it can be readily seen that there is a qualitative difference in the relative order of the lowest-lying excited states in vacuo and in solution: both cases feature the ππ * L a state as the energetically lowest-lying excited state, at 4.93 and 4.77 eV respectively, but while in vacuo S 2 has n O π * nature (5.54 eV, involving the oxygen lone pair of the purinic base), in solution S 2 isthe ππ * L b state, placed at 5.44 eV. This effect has been widely reported in the literature being related to a bathochromic effect (Nakayama et al., 2013), with the n O π * featuring a dipole moment of 5.69 D (3.51 D in vacuo) being less stabilized than the ππ * L b state (7.96 D, 4.92 D in vacuo) as it can be also noticed by looking at their change in dipole moment. The solvent therefore affects the absorption spectra prominently at the FC region by blue-shifting the n O π * state by 0.23 and red-shifting the ππ * L b by 0.33 eV with respect to the gas-phase, a change comparable to those previously reported in other QM/MM studies in solution (Parac et al., 2010;Nakayama et al., 2013). It is worth noting that the oscillator strengths associated to the transitions to the L a and L b states are also affected by the effect of the solvent. Whereas, L a features as the brightest lowest-lying excited state in the gas phase and the L b state presents a slightly smaller oscillator strength (0.158 and 0.145 respectively), in solvent L a and L b present an inverse order in the values for the oscillator strength associated to their transitions (0.09 and 0.17 respectively), being in agreement with the experimental data reported by femtosecond broad-band transient spectroscopy (Karunakaran et al., 2009). The effect of the basis set on the excitation energies has also been studied by employing both 6-31G * and ANO-L 321/21 basis sets. The 6-31G * CASPT2 computations report values of 4.77, 5.44, and 5.77 eV for the L a , L b , and n O π * transitions, respectively, whereas the ANO-L yields absorption energy values of 4.50, 5.10, and 5.71 eV for the L a , L b , and n O π * states respectively, consistent with those reported experimentally (Karunakaran et al., 2009), which place the absorption maximum of L a at 4.50 eV. Table 2 also reports several theoretical approaches found in the literature employing different methods to simulate solvated GMP. As can be seen, more sophisticated DFT/MRCI methods agree qualitatively with the values here reported at the CASPT2 level of theory, yielding values within 0.33 and 0.02 for the L a and L b states respectively with respect to the CASPT2/ANO-L values here obtained, and a slight energetic deviation is also found when comparing to the values computed at a TDDFT level. This small difference present in TDDFT results employing a polarization continuum model (PCM) (see Table 2) could also be due to the fact that (Karunakaran et al., 2009) used a methylated guanine to model the effect of the phosphate group whereas here the phosphate group is explicitly included even if just at the MM level. On overall we can conclude that the absorption values reported here at the CASPT2/ANO-L level are consistent with the experimental data and with the highest-level theoretical estimates present in the literature, thus highlighting the capabilities of the CASSCF/CASPT2 protocol to treat excited states in a balanced manner (Roos et al., 1996) and its usage for mapping photochemical reaction paths (Garavelli, 2006).

Excited-State Evolution
The excited-state evolution is here tracked by means of static approaches through excited-state optimizations and the characterization of the conical intersections representing the crossings among the energetically lowest-lying excited states, ultimate protagonists in the deactivation photo-process. Additionally, the MEPs connecting the initially accessed states and subsequent photoinduced events have been mapped making use of the IRC technique.
The lowest-lying excited state is the ππ * L a state, which is expected to be the main spectroscopic state due to its lowestlying position and relatively large oscillator strength. This state is generally assumed to present an easily accessible CI along its relaxation pathway toward the ground-state characterized by a ring-puckering motion (see Figure 4) widely featured in the DNA/RNA nucleobases (Giussani et al., 2013a) in vacuo. This profile is slightly altered in the presence of polar environments as it has been previously reported for other purine nucleobases (Conti et al., 2010), where a shallow stationary point (L a ) sp arises along the MEP close to the CI with the ground state being placed at 1.15 eV vertically and adiabatically at 3.30 eV from the ground state (see Figure 5). Two different CIs have been characterized in the vicinity of this stationary point, one optimized directly in solvent corresponding to the minimum energy conical intersection (MECI) also reported by Serrano-Andrés  (2008), and another relating to the CI found in the same study along the MEP in vacuo, which we have tentatively named (L a /GS) CI-1 and (L a /GS) CI-2 in the present study, respectively. Both CIs as well as the (L a ) sp present ring-puckering structures in the A6 cycle yielding slight bond-length alterations (C2-N3, N1-C2, N3-C4, and C4-C5) compared to the FC structure and featuring prominent N1-N-C2-N3 dihedral angle distortions at 133 • , 132 • , and 140 • for the (L a /GS) CI-1 , (L a /GS) CI-2 , and (L a ) sp structures, respectively. These distortions are quantitatively different to those reported in vacuo, stressing out the importance of the solvent where the (L a /GS) CI-1 presents a ∼143 • dihedral angle as compared to its 133 • solvated counterpart, together with slightly pronounced bond-length differences as can be seen in Table 1. Both CIs here characterized present very similar structures (see Figure 4) and charge distributions very close to those found in the gas phase, presenting an inversion in their energetic order in solution being (L a /GS) CI-1 the energetically lowest-lying adiabatically at 3.31 eV, (L a /GS) CI-2 being placed 0.3 eV higher in energy and 3.60 eV adiabatically from the FC region. Two different CIs connecting the ππ * L a and ππ * L b states have also been located. Direct CI optimization leads to (L b /L a ) CI-2 , its geometry presenting a deformation near the N9-C8-N7 angle with respect to the ground state. The bond lengths suffer large distortions, observing a shortening of the N1-C6, C5-N7, C4-N3, N3-C2, C6-O, and C2-N bonds, and an increase in the length of the remaining bonds with respect to the FC region (see Figure 4). This CI is placed at 7.62 eV vertically from the FC region, which is higher in energy than both ππ * L a and ππ * L b vertical excitation energies, placed at 4.77 and 5.44 respectively, thus hinting toward a sloped CI profile preventing their nonadiabatic interaction. A second CI, (L b /L a ) CI-1 , obtained along the L b MEP computed within a 4in4 active space to avoid excessive wave function mixing, has been located being placed ∼5 eV adiabatically from the FC region. This CI presents geometrical similarities with its in vacuo counterpart and provide an accessible channel to funnel the initially populated L b state down to L a . Further attempts to optimize the L b state toward a possible minimum have been fruitless due to the large wave function mixing and root flipping problems experienced along the optimization procedure, preventing us to obtain further information on this specific state.
As previously stated, n O π * represents the third energetically wise excited state for the GMP in solvent. The optimized n O π * minimum found in solvent is prominently characterized by an elongation of the C6-O bond distance with respect to the ground state. Table 1 shows the main differences in bond lengths, where the C6-O bond is elongated in this minimum from 1.22 Å at the FC region to 1.41 Å. The minimum of this excited state in solvent is very similar to the one reported in vacuo (Serrano-Andrés et al., 2008), as would be expected given the small effects provided by the solvent on nπ * excited states. Energetically it is placed 3.97 eV adiabatically and 2.67 eV vertically with respect to the ground state (see Figure 6), which constitutes a stabilization of 1.8 eV from its initial value at the FC region.
We have also located the CI between the states n O π * and ππ * L a , (n O /L a ) CI , its geometry presenting a planar structure like the ground state, but showing an elongation of the C6-O bond and a shortening of the C6-C5 bond. We observe similarities between this geometry and that of the CI optimized in vacuo, as they both present the same kind of geometrical distortions given the small solvent effects in the n O π * state, yielding a relative energy of 4.90 eV with respect to the FC region. The CI connecting the n O π * and ππ * L b states, (n O /L b ) CI , has also been located, featuring a planar structure and differences in the bond lengths in the six-member ring, in particular in the differences in the N1-C2, N1-C6 bonds.
The differences observed in the geometries of the excited state minima and of the different CIs characterized both in vacuo and in solvent highlight the importance of the solvent that is considered explicit in our calculations, together with the explicit presence of the ribose and phosphate groups on GMP, providing more accurate estimates to relate to the photoinduced events in the cellular system.  Karunakaran et al. (2009) suggest either bi-or triexponential decay depending on the spectral region measured, providing τ 1 = 0.25 ps, τ 2 = 1.0 ps, and τ 3 = 2.5 ps for the 270-400 nm region in the non-protonated GMP at neutral pH. (Karunakaran et al., 2009) justify these ultrafast signals in terms of TDDFT calculations due to ring-puckering CIs between the ππ * L a and GS states, in accordance to what was previously proposed in vacuo by Serrano-Andrés et al. (2008) on the basis of CASSCF/CASPT2 computations. Molecular dynamics simulations by Serrano-Andrés et al. (2008) proposed that the ultrafast decay from the ππ * L a state could occur already within the first 100 fs, an assumption that has been challenged by Barbatti et al. (2011), pushing the timescale toward the 500 fs mark. (Lan et al., 2009), on the other hand, support a biexponential decay based on their MD simulations where they obtain a faster ∼190 fs and a slower ∼400 fs decays through two different CIs between the ππ * L a and GS states, thus highlighting the complex process under study and the difficulty to simulate it coherently (Mai et al., 2014). The most recent experimental data to our knowledge is based on the photoelectron spectroscopy of GMP both in vacuo and in solution (Chatterley et al., 2014), yielding a biexponential decay of τ 1 = 50 fs and τ 2 = 600 fs in vacuo and τ 1 = 120 fs and τ 2 = 680 fs in solution, which reveals a striking similarity among the two different set of values as they are within experimental error and therefore provide evidence of the negligible role played by the solvent in the photoinduced decay paths present in this system. This biexponential decay is obtained through a 4.66 eV pump, which also suggests the possibility of only probing those kinetic processes undergone after direct ππ * L a population, whereas the time-resolved transient data by Karunakaran et al. (2009) yields a third exponent related to a slower timescale possibly arisen through an initial population of the ππ * L b state.

Photophysics and Photochemistry of GMP: Elucidating Its Intrinsic Photostability
Our present results point toward an ultrafast deactivation along the L a MEP characterized by a ring-puckering motion centered in the C2 atom, which brings the excited-state population to interact non-adiabatically with the ground state, funneling down the population that is further relaxed to the FC FIGURE 5 | CASPT2 energies of the ground (GS) and lowest-lying singlet excited states (ππ * L a , ππ * L b and n O π * ) of the GMP from the FC geometry and along the ππ * L a CASSCF MEP.
FIGURE 6 | CASPT2 energies of the ground (GS) and lowest-lying singlet excited states (ππ* L a , π π* L b and n O π*) of the GMP from the FC geometry and along the n O π * CASSCF MEP.
region. This kind of process has been widely attributed to the DNA/RNA nucleobases and is permanently linked with their intrinsic photostability (Serrano-Andrés and Merchán, 2009) and therefore extends to the photostability of the genomic material. Our calculations in the presence of the solvent and the sugar and phosphate groups reveal a very shallow stationary point at the end of the MEP right before reaching the intersection seam (see Figure 5), which has also been previously documented in other nucleobases at a TDDFT level of theory to be related to a transition state with an imaginary reaction coordinate that drives the system toward the ring-puckering CI (Picconi et al., 2011). This stationary point is explained in terms of the planarity of the ππ * potential energy hypersurfaces of all nucleobases (Giussani et al., 2013a;Improta and Barone, 2014), and is expected to be overcome in order to reach the CIs with the ground state in an ultrafast manner. In contrast, we have shown recently (Conti et al., 2010(Conti et al., , 2015 that the corresponding internal conversion path in adenine is controlled by a larger barrier, which may explain its longer lifetimes. Two different CIs have been located between the ππ * L a and GS states, namely (L a /GS) CI-1 FIGURE 7 | Scheme of the photoinduced processes occurring in solvated GMP upon UV-light irradiation. The energetic values (in eV) and the different crossing points and minima depicted refer to the CASPT2 computations carried out in the present study. The associated time constants marked in yellow have been taken tentatively from the ultrafast pump-probe transient absorption experiments reported by Karunakaran et al. (2009). Three different decay channels have been assigned to the experimental evidence: τ 1 refers to the ultrafast decay path from an initially accessed ππ * L a excitation to the ring-puckering CI with the GS, τ 2 to the initial population of the ππ * L b state and subsequent non-adiabatic population transfer to the L a state finalizing in the CI between L a and GS, and τ 3 that is tentatively assigned mainly to deactivation processes along the L b state (See text for details). and (L a /GS) CI-2 . These two CIs are analogous to those found in vacuo by Serrano-Andrés et al. (2008) but present an energetic inversion due to the embedding effect of the solvent. Both are expected to belong to the same intersection seam given their similar structures, and both are considered to be accessible from the L a stationary point previously characterized. (L a /GS) CI-1 , being the lowest-lying energy wise is expected to embody the fastest decay route, relating to the τ 1 = 0.25 ps recorded through timeresolved transient and τ 1 = 120 fs in time-resolved photoelectron spectroscopy experiments. This assignment is analogous to that previously reported for 9H-Guanine in vacuo, and supports recent experiments reporting analogous excited-state decays for GMP in gas-phase and in solution (Chatterley et al., 2014). The second conical intersection with the ground state, (L a /GS) CI-2 , might be responsible for the second exponential measured at τ 2 = 1.0 ps and τ 2 = 680 fs in time-resolved transient and photoelectron spectroscopy, respectively, given its higher-lying energetic position and a relatively small barrier of ∼0.3 eV to be overcome in order to access it once reaching the intersection seam. Nevertheless, the existence of another CI between the L a and GS states not characterized in the present contribution could better explain that second lifetime, which will be related to the L a surface as it has been recently reported experimentally (Chatterley et al., 2014). These assignments are in line with the MD studies by Lan et al. (2009), reporting two different decay routes with their associated timescales to two distinct CIs between the ππ * L a and the ground state, and are in line with other theoretical CASPT2 and TDDFT studies that point toward the L a state as the responsible for both ultrafast decay timescales.
The ππ * L b state is predicted to be involved in the photoinduced events being related possibly only to the longest-lived τ 3 . This is explained in terms of an initial population of the L b state, which presents a sizable oscillator strength, and subsequent funneling of the population toward the L a state mediated by the (L b /L a ) CI-1 , placed at ∼5 eV along the L b relaxation pathway. The second CI characterized between L b and L a states, (L b /L a ) CI-2 , is expected to be irrelevant to the photo-process due to its highlying energetic position preventing its accessibility. The lone-pair excited state, n O π * , is blue-shifted in solution as has been already mentioned above. Its elevated vertical absorption energy prevents it to be one of the main spectroscopic states, yet its close-lying position to the absorbing ππ * L b state facilitates a possible population of this state and subsequent relaxation toward its minimum, (n O π * ) min . This minimum is placed at 3.97 eV adiabatically and 2.67 eV vertically from the GS, and could be partly responsible of the longest-lived signal reported experimentally at τ 3 = 2.5 ps or at τ 3 = 167 ps at low pH, given that such nπ * -mediated processes have been already characterized experimentally to be close to the ∼100 ps timescale on pyrimidines both in vacuo and in solution (Hare et al., 2007).
The present study elucidates the photoinduced events in GMP in terms of an ultrafast decay along the main spectroscopic and initially accessed ππ * L a state characterized by a ring-puckering motion, which would cover the experimental timescales τ 1 and τ 2 through different CIs with the ground state, whereas the longest-lived component would be attributed to the decay routes mediated through the ππ * L b and, to a minor extent, to the n O π * state.

Conclusions
The present study encompasses a photophysical and photochemical appraisal of water-solvated GMP by means of theoretical multireference perturbation theory QM/MM techniques. An initial MD simulation has been carried out in order to characterize the geometrical parameters of the FC region. The vertical excitation energies have been computed and compared to recent data found in the literature and to the results obtained in vacuo, highlighting the importance of the environment yielding qualitative differences for the ππ * L a and ππ * L b states being red-shifted and for the n O π * state being blue-shifted as compared to their gas-phase counterpart. The ππ * L a state is predicted to be the main spectroscopic state driving the ultrafast deactivation processes occurring in GMP upon UV-light irradiation based on a ring-puckering motion that enhances its non-adiabatic interaction with the ground state in a radiationless manner. A shallow stationary point toward the end of the ππ * L a MEP has been characterized, together with two different CIs with the ground state that help rationalize the two fastest decay times measured experimentally. Upon initial L b absorption, two CIs between the ππ * L b and L a states have also been located, one of them along the L b decay path pointing toward a population funneling down to the L a state and another being too high in energy to contribute prominently in the photo-process. The CIs connecting the n O π * state and the ππ * L b and L a states have also been characterized along its Frontiers in Chemistry | www.frontiersin.org relaxation route, yielding a minimum in the n O π * state expected to emit vertically at ∼2.7 eV. Both ππ * L b and n O π * are suggested to contribute to the longest-lived experimental timescale, the latter providing a lesser contribution given the relatively fast kinetic timescale and the long-lived timescales expected in nπ * fluorescent states. Upcoming QM/MM dynamics simulations are expected to shed some more light on the photoinduced events occurring in water-solvated GMP and its specific decay timescales in order to provide a more specific molecular counterpart to the experiment and better explain the photochemical and photophysical processes resulting in the intrinsic stability of the genomic material.