Ultrafast Nuclear Dynamics of Photoexcited Guanosine-5

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Ultrafast Nuclear Dynamics of Photoexcited Guanosine 5#-Monophosphate in Three Singlet States Sayan Mondal, and Mrinalini Puranik J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05735 • Publication Date (Web): 27 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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The Journal of Physical Chemistry

Ultrafast Nuclear Dynamics of Photoexcited Guanosine 5ʹmonophosphate in three Singlet States Sayan Mondal and Mrinalini Puranik* Indian Institute of Science Education and Research, Pune‒ 411008, India

*Corresponding Author Address: Indian Institute of Science Education and Research Dr. Homi Bhabha Road, Pashan, Pune˗411 008, India Phone: +91˗7350694600 E˗mail: [email protected] Current address: Unilever Research and Development Centre, 64 Whitefield, Bangalore 560066, India

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Abstract We report measurement of resonance Raman (RR) spectra of 5′-guanosine monophosphate (GMP), a DNA nucleotide at excitation wavelengths throughout its ππ* absorption band (Bb) in 210-230 nm range. From these data, we constructed wavelength-dependent Raman intensity excitation profiles (REP) for all observed modes. These profiles and the absorption spectrum were then modeled using self-consistent simulations based on the time-dependent wave packet propagation (TDWP) formalism. We inferred the initial structural dynamics of GMP immediately after photoexcitation in terms of dimensionless displacements. The simulations also provide linewidth broadening parameters that in turn report on the time-scale of dynamics. We compared deduced structural changes in the purine ring upon photoabsorption into the Bb state with those deduced for the two lowest lying ππ* (La and Lb at 280 and 248 nm respectively) excited states of GMP. We find that excitation to Lb state lengthens C6‒N1 and C2=N3 bonds which lie along the formation coordinate of various oxidative adducts but Bb excitation does not. We also find that photoabsorption by Bb state weakens C8‒N9 bond and thus might assist imidazole ring opening via cleavage of the same bond. Electronic excitation to different ππ* states of the guanine chromophore results in contrasting structural changes; while absorption by La and Lb state induces expansion of pyrimidine and contraction of imidazole rings, Bb excitation results in overall shrinkage of both the rings. Computed absolute changes in internal coordinates imply that photoexcitation to any of the three singlet states of GMP does not lead directly to the formation of a cation radical of guanine.


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Introduction DNA and RNA store genetic information, and this is translated to synthesize specific proteins in organisms. The accuracy of the information depends on the chemical identity and properties of the nucleobases within DNA and RNA. These molecules, therefore, have to be robust against a variety of natural modifying agents such as reactive molecules in the cellular milieu and sunlight among others. The five natural nucleobases are strong absorbers of ultraviolet (UV) light because of which our genome is continually prone to UV-radiation induced photodamage. The genome remains protected however because of the remarkable efficiency with which these nucleobases dissipate excess energy deposited by radiation. Experiments have shown that natural DNA and RNA bases spend less than ~1 ps in the first singlet ππ* excited state following UV photoexcitation whether in isolation, as nucleosides or nucleotides.1–7 Computational modeling of excited state potential energy surface (PES)8–11 and photodynamical simulations12–16 have been used to investigate relaxation pathways of these nucleobases. These studies revealed that ultrafast internal conversion (IC) from the singlet excited to the ground state is driven by out-ofplane distortions of the molecule. These distortions lead the molecules towards conical intersections (CI) between different PESs, and thus quickly returning the molecules to the less reactive ground state. This efficient deactivation by efficiently dumping absorbed UV energy makes them more photoresistive than other analogous molecules. It is argued that this intrinsic resistance against photodamage may have contributed to their evolutionary selection as the building blocks of our genetic code.17 Despite their robust photoprotection, natural nucleobases do undergo photodamage by UV radiation and reactive oxygen species (ROS). Guanosine-5′-monophosphate (GMP), the nucleoside of Gua (Figure 1) is particularly important because of its ability to form a lesion through one electron oxidation. The product of this oxidation ultimately leads to a mutation in the genome via misreading of DNA by polymerase enzymes during replication. Photochemical damage occurs following distortions of the molecule in the excited state after absorption of a photon. The changes in the structure of the molecule due to its excess energy make it highly reactive. To understand the photoreactivity of GMP upon UV irradiation, the photophysics of the first two singlet states (La and Lb) has been extensively studied using femtosecond transient absorption (fsTA),18,19 UV pump mid-IR probe,20,21 picosecond time resolved IR (psTRIR),22 fs time resolved fluorescence,1,23,24 resonance Raman (RR) spectroscopy25 and dynamical 3 ACS Paragon Plus Environment


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simulations.26–28 When GMP is excited into La (280 nm) state it returns to S0 via ultrafast IC within one ps. On the other hand, when excited to the next bright state, Lb, at 252 nm, it is followed by first an ultrafast IC from Lb to La within 100 fs followed by return to S0. RR spectroscopy of 2′-deoxyguanosine (2′-dG) has revealed that three in-plane ring stretching vibrational modes at 1366, 1490 and 1573 cm-1 couple these two close-lying electronic states providing the path for the energy transfer between the two states.25 Apart from the La and Lb excited states, GMP has other stronger ππ* transitions. These excited states are located in the UV-C region around 200 nm (called Bb hereafter) and ~187 nm respectively.29 Since photoabsorption in this region is more efficient, it is important to understand the mechanisms through which the resulting excited states dissipate energy for an overall picture of the photophysics of GMP. It is also important to understand UV-C response of nucleobases from an evolutionary perspective because the flux of solar radiation was several folds higher in the prebiotic era than at present value.30,31 Effective decay of UV-C photon energy would have been instrumental for the continued existence of our genetic materials. However, our understanding of the effect of UV-C radiation on nucleobases is limited. Ultrafast spectroscopic techniques that are routinely applied to investigate photodynamics of excited states within 260 nm absorption band are not readily applicable to studies involving high energetic electronic states. The uncertainty principle couples spectral width and time resolution (∆E × ∆t ~ ħ) putting inherent limits on direct femtosecond time-resolved measurements of these highenergy excited states. Resonance Raman (RR) intensity analysis is an approach that provides a way around this by using spectral broadening to infer timescales of dynamics.32,3334,35 This approach also yields initial structural dynamics of molecules along their vibrational normal modes34–37. Frequencies in a vibrational Raman spectrum report on the strength of bonds between atoms thus the molecular ground state, S0. The intensity of RR bands, on the other hand, is determined by the overlap between the ground state and the excited state along the corresponding normal mode. In the Frank-Condon (FC) region, the intensity is proportional to the square of the slope of the PES along different vibrational coordinates of the resonant vibronic state. The ground state structure and the normal mode descriptions are available from computational modeling, and the normal Raman intensity can be computed. Computing the resonance 4 ACS Paragon Plus Environment

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enhancement of the intensities requires knowledge of additional parameters such as the transition dipole moment between the ground and the resonance state, the separation between the lowest vibrational states of the ground and resonant state, and the change in the excited state structure with respect to the ground state, etc. Starting with an estimate of these values from experimental or computational sources, it is possible to make an iterative, self-consistent fit to the experimental measured Raman intensities at multiple excitation wavelengths by varying the parameters of the system. Conversely, these the final iterative fit yields the parameters that characterize the excited state. Thus, such a simulation of the intensities yields the instantaneous structural changes of the molecule in the excited state in comparison with that in ground state. We recently demonstrated an application of this technique to 6-chloroguanine following photoexcitation within 210 nm absorption band.38 Herein we report measurement of the Raman intensity profiles of the parent nucleobase, GMP in 210-230 nm region within Bb absorption band. We combined quantitative Raman intensity measurements with extensive computational modeling of solvated GMP. We simulated the measured intensity profiles with using the semiclassical approach of Lee and Heller.32,34 Put together, these studies reveal the initial excited state dynamics of GMP upon deep UV excitation. Strong resonance enhancement of in-plane vibrational modes makes this technique suitable for interrogating GMP in different ππ* states.25,39–45 We simulate these REPs using LeeHeller’s time-dependent wave packet propagation (TDWP) formalism32 and derive parameters describing nuclear dynamics of GMP within 50 fs of photoexcitation. We obtain dimensionless displacements (∆) along each of the FC active vibrational coordinates of GMP, and determine structural distortions of the purine ring there from. Additionally, we also determine amplitude and time scale of the inertial component of solvation employing Brownian oscillator model.46 Total linewidth broadening that contributes to both REPs and diffuse absorption spectra are partitioned into fast and dynamics homogeneous, and slow and static inhomogeneous components. We present a comprehensive comparison of photo-initiated sub-50 fs dynamics of GMP in its three singlet excited states. We compare current results with the previously reported early time structural and solvation dynamics of 6-ClG subsequent to photoexcitation within 210-230 nm wavelength range.38 6-ClG is a guanine analogue in which the oxygen in the carbonyl group is substituted 5 ACS Paragon Plus Environment


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with the heavier and less electronegative chlorine atom. With a direct comparison of excited state dynamical parameters in case of GMP and 6-ClG, we determine the role of substituent at the C6 site governing structural dynamics and photoreactivity of guanine chromophore. We find that change in the exocyclic substituent at various sites on purine and pyrimidine rings not only perturbs their ground state structure and also affects photophysics on excited state PES.

2. Material and Methods 2.1 Determination of RR cross-section GMP (Guanosine-5’-monophosphate, 99%) was purchased from Fluka and used without further purification. Accounts of experimental methods for measuring REP in 210-230 nm UV excitations have been described elsewhere.38,47 In brief, 4th harmonic of a Ti-sapphire laser of 25 nanosecond pulse width and of 1 KHz repetition rate (Photonics Industries, Bohemia, NY) producing fundamental IR light within 840˗920 nm was used as the excitation source for RR measurements. A 135o backscattering geometry was used for optimal collection of scattered radiation through a monochromator (SPEX 1250M, SPEX Industries, Edison, NJ) of 1.25m focal length equipped with 3600 grooves/mm holographic grating. The dispersed photons were detected on a charge couple device (CCD) having 1024 x 256 pixels and cooled with liquid N2. Typical laser power on sample was less than ~ 600 µw. For optimal signal collection and good resolution; entrance slit of spectrometer was set to 300 µm. GMP, dissolved in milliQ water (1 mM, pH 6.8) in a quartz tube, spinning along its axis has been used for RR measurements. Sodium perchlorate (0.5 M) was used as an internal intensity standard (IS). Spectra of three freshly prepared GMP samples were recorded at any excitation wavelength. No photodamage was observed as confirmed by comparison of spectra recorded at the first and last minutes of a total accumulation of 10 minutes. Recorded RR spectra were calibrated using positions of known bands positions of organic solvents e.g. acetonitrile, isopropanol, cyclohexane and dimethyl formamide of HPLC grade, which were purchased from Ranchem Chemicals (Shanghai, China) and Sigma˗Aldrich (St. Louis, MO). Sample bands were deconvoluted with Lorentzian functions and integrated area under each band was used as intensity. Absolute RR cross-section of all GMP bands were determined using the following equation,35,48


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σ =

∂σ 8π 1 + 2ρ C I λ Kλ , λ , 1 3 1 + ρ C ∂Ω ∥

where, ρ is the depolarization (dp) ratio of the Raman bands of GMP. !" and !# are the intensities

of the band corresponding to the Nth mode of GMP and the band of perchlorate at 932 cm-1,

respectively. CN and CS are concentrations of GMP and internal intensity standard respectively. (∂σs/∂Ω) || + ┴ is the total differential cross-section of a band with respect to the 932 cm˗1 band of sodium perchlorate.49 Response function of the monochromator and detector, S(λ) = D(λ)/Τ(λ) was used to correct sample band intensity (Icorrected) for the spectral sensitivity of the spectrometer.34 Self-absorption of resonance Raman scattered photons lying within the absorption band of the molecule is also corrected.50,51 Depolarization ratios are measured using a method described elsewhere38,47 and validated by measuring the same for standard organic solvents and comparing with values obtained by other groups.52 (Table S1 of the Supporting Information) 2.2 Simulation of RR cross-section The resonance Raman excitation profile and absorption spectra of GMP within the Bb absorption band are simulated using self-consistent time-dependent wave packet (TDWP) formalism,32,33 Raman cross-section $% &' =

8π( E* E+ M( 9ℏ/ c ( θ√2π =

=

3 dE exp 8− :

E − E: ; < 2θ;

iE+ − E: t ΔG exp−iωG t − 1 × ?3 exp 8 − g CDE t< ℏ √2 :

× I exp J− KQR

Δ;K 2

Absorption cross-section

=

;

L1 − expM−iωK tNOP dt? , 2

=

E − E: ; iE+ − E: t σS E+ = 3 dE exp 8− < × 3 exp 8 − g CDE t< ; ; 2θ ℏ 6ℏ V c θ√2π 4π( E+ M ;

:

× I exp J− KQR

Δ;K 2

W=

L1 − expM−iωK tNOP dt , 3



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where, EL is the incident radiation, and ES is the energy of scattered Raman photons, n represnts the refractive index of the medium, M is the transition dipole moment strength in A, ωj is the frequency of the jth normal mode, and E0 is the zero-zero transition energy. The static inhomogeneous broadening (θ) is introduced in both equations as a phenomenological broadening parameter in the expression of the normalized Gaussian distribution of zero-zero energies about an average value of E0. θ represents solvated GMP population of slightly different micro-environments, which are static in nature within the time scale of the Raman process. ∆j is the difference between the ground and excited state minima along the jth normal mode in ground state and is expressed as a ‘dimensionless” quantity. The solvent induced homogeneous broadening that arises from instantaneous solutesolvent interactions upon photoabsorption by the solute is modeled according to the Mukamel and Myers Brownian oscillator model.46,53 This effect is introduced as a function, gsolv, describing solvent induced dephasing originating from interaction between solute transition dipole and bath coordinate and described previously.38,47 (See Equation S1 in Supporting Information) The total internal reorganization energy of the photo excited GMP is determined by the

; expression, XYZ[ = ∑*"W/ ]QR Δ] ℏωK /2. These ∆j’s can be converted to geometric parameters such

as changes in internal coordinate corresponding to the jth normal mode (δj) using54 W

e

δK = 5.8065 ∑K AKd ωK f ΔK , 4 where ωj is the frequency of jth normal mode in cm-1 and Aji is the jith element of the inverse of the matrix that projects internal coordinates onto the normal mode coordinate. Extinction coefficient (ε in L mol-1 cm-1) and absorption cross-section (σA in Å2 molecule1

) of GMP is determined. (See Equation S2 in Supporting Information), and obtained value of ε

(Table 1) agrees with published results.56 Initial guesses for the ∆’s along different normal coordinates were obtained from relative intensities of all the twelve RR bands with respect to the band at 1365 cm-1 at 210 nm excitation wavelength. The transition dipole moment strength of 0.96 Å was derived from deconvolution of 8 ACS Paragon Plus Environment

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the experimental absorption spectra by Gaussian line shape (Figure 2, Panel b, bottom). Initially, zero-zero transition energy was estimated such that the red edge of simulated absorption and REPs of all modes agreed with experimental values. Values of the parameters were then iteratively optimized by using a self-consistent procedure to fit the REP and absorption spectrum simultaneously with experimentally measured spectra.55 The quality of the fit was determined by visual inspection and later by varying each parameter by 10 % of its best fit value. The simulation is performed using a commercial MATLAB package (The MathWorks, Natick, MA) 2.3 Determination of absorption cross-section 2.4 Quantum chemical calculations All computations were performed with Gaussian 09 software suite.57 GMP in aqueous solution is modeled as 9-methylguanine (9-meG) in complex with six explicit water molecules, 9meG•6H2O. In addition, polarizable continuum model of solvation was employed to take into account the bulk dielectric environment of water.58 Six water molecules were strategically placed around isolated 9-meG within hydrogen bond contacts (Figure 1) as reported for GMP in other studies.18 Energy minimized molecular structure on S0 state, harmonic vibrational frequencies and associated normal mode vectors, and excited state transition dipole moments are computed using B3LYP hybrid functional.59,60 Geometry optimization of S0 state was performed without any symmetry constraints. Pople type basis sets that are augmented with diffuse and polarization functions, 6-311+G(2d,p) was used for reliable description of electronic excited states. Excited state energies were computed as vertical transitions on the S0 geometry using linear response formalism of time˗dependent DFT (TD-DFT) method.61,62 Coordinates of the optimized structure of 9-meG•6H2O complex is described in Table S2 of the Supporting Information. The matrix Aji of Equation 4 is obtained from computation of normal modes on ground state equilibrium structure.

3. Results and Discussions 3.1 Electronic structure of Bb state UVRR spectra of the Gua chromophore have been previously reported by several groups with Raman excitations lying within Bb, La and Lb absorption bands of GMP and 2′-deoxy GMP (dGMP) and different N9 substituted forms.25,39–45,63,64 Figure 3 shows the spectrum of GMP we 9 ACS Paragon Plus Environment


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obtained with a laser light of wavelength 212 nm. Highest enhancement is observed for triene modes at 1365 and 1580 cm-1 and of the carbonyl stretching mode at 1689 cm-1 in agreement with previous measurements.40,41 Resonance enhanced modes in each of the three electronic states (Bb, La and Lb) are primarily in-plane ring stretching vibrations due to their strong ππ* character. Semi-empirically derived transition monopoles for ππ∗ transitions of Gua have suggested that excitation to Bb state would result in electron delocalization over both rings of Gua,65 later confirmed by complete active space self-consistent field (CASSCF) calculation.66 Our TD-DFT calculations (Table 1) also describe this state as consisting of pure ππ* character, with 77% of H‒1→L, one electron configuration and delocalized over both the rings (Figure 2a). (See Table S3 of the Supporting Information for complete state assignment) This description agrees with CASSCF computed configuration, H‒1→L (51%) for Bb state of N9H-Gua.66 Singlet excitation energies of Gua were calculated by several authors in gas phase,11,65,67,68 and in solution.18,66,69–73 Comparison with published excitation energies shows that magnitudes of computed vertical excitation energies of Gua vary depending on what level of theory, basis sets and solvation models are employed.74 (Table 1) Using TD-B3LYP method to obtain the solvated structure of 9-meG•6H2O complex, we have found good agreement between computed vertical transition energies of four ππ* electronic states of GMP and experimental values. (Table 1). 3.2 RR Characterization of Bb state Figure 4 demonstrates variation in the intensity of RR active modes of GMP within Bb absorption band within 210-230 nm as a function of excitation wavelengths. Experimental REPs show that as the wavelength of excitation light is tuned from 210 nm towards 230 nm all modes loose intensity. Only the band at 1486 cm-1 shows the opposite trend. This loss of intensity indicates coupling of this mode with the low lying Lb state in addition to the Bb state we are investigating. The Lb state has been extensively investigated by Loppnow and coworkers in 2′deoxyguanosine (2′-dG) with Raman intensity measurements in the 244-290 nm range.25 They found that initial nuclear dynamics of photoexcited 2′-dG lies along the modes at 1490, 1580, and 1689 cm-1 in La excited state, and 1321,1490, 1580, and 1607 cm-1 in Lb excited state. From the spectrum in Figure 4, we anticipate that primary structural dynamics within Bb absorption band would occur along the modes corresponding to these bands with high intensities: 1177, 1365, 1580 and 1689 cm-1. 10 ACS Paragon Plus Environment

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Absorption cross-section and constructed REPs of RR bands of GMP are shown in Figure 5 and 6. Intensities of six resonant modes at 1076, 1177, 1210, 1255, 1365, 1413, 1540 and 1689 cm˗1 of GMP follow the red edge of the Bb state. Four modes at 867, 1046, 1580 and 1607 cm˗1 gain additional intensity at higher excitation wavelengths, above 222 nm. The blue edge of the Lb absorption band extends to 222nm. Thus, red edge of the current measurement band for the REP, 223˗230 nm overlaps with the blue edge of the Lb band. Thus the deviation in the vibrational band intensity indicates that the corresponding normal modes couple the Bb and Lb electronic states and are FC active in them. A similar coupling has been seen between the Lb and La states in 2′-deoxyguanosine (dGuo) where three bands viz. 1490, 1573 and 1600 cm˗1 show deviation in intensity and the corresponding normal modes couple these states.25 Interference between these two states had not been anticipated from apparently well-separated bands in absorption spectra. Change in delocalized π electron density (Figure 2a) over the whole purine ring permits enhancement of ring modes at 1076, 1177 and 1365 cm˗1 arising from stretching (str.) of pyrimidine, imidazole bonds, and triene coordinates (along N3‒C4‒C5‒N7) respectively. Computed normal mode description shows that significant amount of NH2 rocking motion is involved with two in-plane ring modes at 1076 and 1177 cm˗1. Amino scissoring vibration appearing at 1607 cm-1 has moderate intensity with excitation at 212 nm wavelength. The molecular orbitals (Figure 2a) involved in Bb transition show that the π electrons, localized on NH2 moiety do not take part in the electronic transition, but are involved in another dipoleallowed ππ* transition (state S11 in Table 1). Thus enhancement of this vibration is expected to originate from the contribution of the later electronic state via Albrecht’s B term.75 The FC coupling of two modes at 1580 and 1607 cm˗1 with Lb state is supported by previous work as well.25 The fundamental mode responsible for the band at 1580 cm˗1 originates from NH2 scissoring (sci.) and C‒N str. (C2‒N3 + C4‒C5 and N7‒C8 str.) while the band at 1607 cm˗1 is ascribed to pure exocyclic NH2 sci. motion. From REPs of 2′-dG measured within La and Lb absorption bands,25 it is evident that though both of these RR modes gain intensity in Lb state, amino sci. vibration at 1607 cm˗1 gradually decouples from La state. Measured depolarization (dp) ratios (ρ) of RR bands of GMP at 210 nm excitation wavelength are tabulated in Table S4 of the Supporting Information, and depolarized and polarized spectra are shown in Figure S1 of the Supporting Information. A value of ρ = 0.18 for 11 ACS Paragon Plus Environment


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the 1607 cm˗1 mode at 210 nm excitation indicates interference from the red edge of the S11 transition. Thus, the effective cross-section of this mode must result from interference between Bb and the next allowed transition. The shape of the REP of the band at 1365 cm˗1 suggests plausible coupling with both of the Lb and S11 states. This mode is delocalized on several purine N‒C internal coordinates that suffer changes in bond order during photoexcitation to any these states. A dp ratio value of 0.17 of this band at 210 nm excitation implies interference from Bb band, located on the blue side of our probe wavelengths. Our TD-DFT computation also predicts a bright state (S11) on the blue side of the Bb band. We measured the dp ratio at 210 nm excitation which is near the maximum of the Bb band (Figure 2) and obtained a value of ρ close to 0.33 for the two modes 1486 and 1689 cm˗1. This suggests that their RR enhancement is due to sole contribution from the Bb state. The REP of the carbonyl stretching vibration at 1689 cm-1 follows the red edge of the Bb absorption band faithfully. This mode does not couple to the Lb electronic state; a fact also supported by the very low intensity at excitation wavelengths within the Lb band at 244 and 257 nm.25 The carbonyl stretching coordinate couples with the lowest singlet excited state La as expected from the direction of the transition dipole moment and reported RR enhancement of this mode at 290 nm Raman excitation. Tinoco and co-workers have suggested that the enhancement of the carbonyl str. mode at 1689 cm-1 with 200 nm excitation is probably originating from the high lying S11 transition at ~190 nm because the Bb monopole contribution is not localized on this part of the molecule.65 We find no evidence of this as our measured dp ratio of 0.30 with 210 nm excitation indicates that major contribution is from a single excited state. 3.3 Geometrical changes upon photo-excitation The simulated absorption and REPs for twelve resonant modes of GMP are shown in Figure 5 and 6, and the best fitted parameters are depicted in Table 2. Parameters corresponding to La and Lb excited states of 2′-dG25 and the Bb excited state of 6-chloroguanine (6-ClG)38 are also tabulated for comparison. A value of depolarization ratio close to 0.33 for intense bands of GMP at 210 nm excitation indicates that the Bb state is primarily responsible for the FC activity of these RR active modes. Apart from a low intensity mode at 679 cm-1, measured REP of all 12 RR active modes of GMP have been modeled using a single excited state using the time 12 ACS Paragon Plus Environment

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dependent theory of RR scattering. Although we have obtained a good fit between experimental and simulated cross-sections, deviations at places clearly suggest the presence of interference from high and low lying S11 and Lb states respectively as discussed at length above. The breadth of the diffuse absorption band shape is determined by the relative contribution of ∆s of all RR active modes. Thus, though measured REPs are on the red edge of the Bb absorption band and do not cover both sides of the Bb transition, a correctly deconvoluted absorption spectrum (Figure 2) guarantees reliable estimation of ∆s. The inverse of solvent correlation time, (Λ=1/2πcτ) is insensitive to RR cross-section and absorption spectra at a specific wavelength but depends on the overall shape of the REP. As complete REP is not available on both sides of the absorption peak, Λ is fixed at 250 cm-1, a value close to what is estimated for similar sized molecules as 6-ClG in Bb and tryptophan in Bb electronic states. In the current study, the effect of this parameter is not tested on simulated RR cross-section. Structural distortions along different internal coordinates following photoexcitation to all three singlet excited states of GMP are determined using Equation 4 from best fitted ∆s and are described in Table 3. Following photoexcitation to the La state, both the imidazole and the pyrimidine rings undergo similar amounts of absolute distortion (0.09 and 0.08 Å respectively) with respect to their ground state structures. However, after taking into account the sign of these distortions, it is found that the imidazole ring decreases in size through a major decrease in N7=C8 and N9–C4 bond lengths while in pyrimidine, the C5–C6 bond suffers maximum distortion (‒ 0.037 Å ). Because of comparatively high transition strength, photoexcitation into the Lb state leads to more absolute amount of distortions in both the rings than those corresponding to the La state. The pyrimidine ring experiences more distortion than imidazole and expands through N3– C4, C6–N1, and C2=N3 bonds. Photoabsorption by the Bb state of GMP produces contrasting effects from those of the Lb state; inducing an overall contraction of both the pyrimidine and the imidazole rings by 0.22 and 0.04 Å respectively. Imidazole distortion occurs through weakening and strengthening of the C8– N9 and the N9–C4 bonds by 0.07 and 0.07 Å respectively. The Lb transition couples with the imidazole ring via C5–N7 and N7=C8 internal coordinates and the Bb state does it through C8–N9 and N9–C4 bonds. The pyrimidine ring specifically couples with the Bb transition via the N1–C2 str. coordinate, but not with any of the other two electronic states. For both Bb and Lb 13 ACS Paragon Plus Environment


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photoexcitation, atomic bonds localized on the triene coordinate and the C6–N1 bond undergo major distortions. Lengthening of the C2–N2 bond by maximum amount (0.06 Å) in case of the Lb state implies the presence of strong coupling of this transition with the exocyclic amino group. This fact is in agreement with an enhancement of the band at 1607 cm-1 that arises primarily from NH2 scissoring with excitation line within the Lb absorption band.25 Two exocyclic bonds on pyrimidine and imidazole rings i.e., C6=O and N9–Cmethyl are found to be strengthened due to charge redistribution following photoexcitation to all three singlet states. Their absolute magnitude in La state is the lowest owing to comparatively low transition strengths of this state among all three. This finding indicates that UV excitation of GMP does not induce geometrical changes that coincide with the photo-dissociation coordinate of either oxo or methyl (ribophosphate) groups. Chloro substitution at the place of oxygen in the Gua chromophore causes dramatic changes in terms of distortion of both the rings; imidazole gets expanded with a concurrent contraction of the pyrimidine ring in 6-chloroguanine (6‒ClG). Two component bonds of the triene coordinate, C5=C6 and C4–C5, do not couple with the Bb state of 6‒ClG as in GMP. Though overall magnitude of distortion is much less in 6-ClG than in GMP due to absorption by Bb state, the C6−Cl bond is found to be shortened by 0.06 Å: a value similar to that of the C6=O bond in GMP. 3.4 Implication on adduct formation For Gua residues in DNA, two major types of photochemical damages can occur: (i) formation of apurinic or abasic (AP) sites through cleavage of the glycosidic bond at N9; and (ii) oxidative modification leading to the formation of formamidopyrimidine and 8-oxo-7,8-dihydro2′-deoxyguanosine (8‒oxodG) via cleavage of the C8–N9 bond, and oxidation at C8 by a hydroxyl radical respectively.76 In addition, oxazolone and its precursor imidazolone77,78 are also oxidation products of dG. 8-oxodG is 1000 times more reactive than dG,79 and forms oxazolone80 as a secondary product upon oxidation by various oxidizing agents. However, it is important to note that none of these adducts are results of direct irradiation of Gua by UV light. These adducts form through oxidation of dG and 8‒oxodG by oxidants such as peroxynitrite81 and various reactive oxygen species (ROS). The amount and the direction of photo-induced instantaneous structural changes can point out if the primary reaction coordinates of formation of any of the mentioned adducts coincide with initial structural dynamics of GMP. 14 ACS Paragon Plus Environment

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Photoabsorption by all three dipole-allowed states triggers weakening of the C8–N9 bond, specifically by 0.07 Å in case of Bb. This indicates initial structural dynamics of GMP lie along the formation coordinate of the formamidopyrimidine adduct as predicted by Yazbi et al.25 In case of all three electronic transitions, the N9‒Cmethyl bond gets strengthened, indicating that photoexcitation alone does not lead to direct cleavage of the glycosidic bond. Decomposition of the pyrimidine ring at C6 site through cleavage of the C6‒N1 bond can lead to formation of another lesion, oxazolone. Cleavage of C2=N3 with a following breakage of the C6‒N1 bond leads to formation of a carbodiimide (HNCNH) fragment in collision induced dissociation (CID) of Gua in gas phase.82 Loss of an -NH3 group as the dissociation product of guanine also happens via cleavage of the C6‒N1 bond.83,84 We find that photoexcitation within the Lb absorption band weakens both of C2=N3 and C6‒N1 bonds which are the primary steps for formation of mentioned lesions. On the contrary, excitation to the higher energetic Bb state does not cause any of these structural distortions and thus does not directly assist the decomposition of the pyrimidine ring at the C6 site. Quantum chemical calculations show that during formation of a cation radical of 9-meG (9-meG+•) by the loss of an electron, the purine ring suffers most distortion along the hexatriene like motif along N2–C2=N3–C4=C5–N7=C8–N9 bonds. Structure of radical of 9-meG (Fig S2 and Table S4 of the Supporting Information) and distortions of bonds with respect to neutral species computed by us is in agreement with previously reported results on N9H-Gua and its cation radical.82,85 (Table S5 of the Supporting Information) 9-meG+• forms by removal of an electron from the hexatriene bond sequence in the neutral molecule. As the lone pair of the amino group at the C2 site contributes significantly in conjugation with this sequence, the C2‒N2 bond gets shortened by 0.05 Å along with a partial planarization of the amino group. All these bonds experience 0.03‒0.05 Å change in length with alternating directions, starting with lengthening of C2=N3 bond (Table S5 of the Supporting Information). The initial structural changes following photoexcitation to any of the GMP excited states do not lead to a decrease in the C2‒N2 bond length, but instead cause an increase of 0.01 and 0.06 Å in La and Lb respectively, whereas Bb excitation does not perturb this exocyclic bond at all. In fact, in La and Bb states, distortions in all these bonds lie in exactly the opposite direction compared to the structural changes that must occur to form Gua+• from neutral Gua. Thus we conclude that direct photoexcitation to any of the three singlet electronic states of GMP does not induce instantaneous distortions that may lie 15 ACS Paragon Plus Environment


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along formation coordinate of its cation radical. However, possibility of existence of indirect pathways, leading to formation of a cation radical of guanine cannot be completely ruled out, as RR intensity analysis can probe the excited PES only a few tens of fs after photoexcitation. Other time-resolved spectroscopic techniques need to be employed to examine the excited state PES further, and to firmly establish our conclusion. 3.5 Internal reorganization, solvation and linewidth broadening mechanisms Experimental and computed wavenumbers, vibrational mode assignment, potential energy distribution (PED), and estimated mode-specific internal reorganization energies for five key RR active modes of GMP are described in Table 4. (See Table S6 of the Supporting Information for the complete assignment) Total internal reorganization energy (λint) of GMP determined from best fitted ∆’s is 995 cm-1 comprises of 28% of the total reorganization energy, 3545 cm-1. A similar value of λint (904 cm-1) has been obtained for 2′-dG in Lb state (see Table 2). Though total reorganization energies are of comparable magnitudes, their mode specific contributions result in different amounts of changes in internal coordinates as discussed in preceding sections. Unlike in GMP, a relatively low value of λint, 112 cm-1 was obtained for 6ClG upon excitation to Bb state.(see Table 2) The chlorine atom at the 6th position of purine not only restores conjugation of the pyrimidine ring of GMP, and causes a ~ 30 nm red shift of the La state but also alters overall electronic structure. Introduction of multiple close-lying states near intense Bb band (~220 nm) on 6-ClG eventually results in structural distortions that are minor compared to those GMP. Steady state fluorescence quantum yield of natural nucleobases, their nucleotides, and nucleosides at room temperature are within 0.68‒1.54 × 10-4 and have peaks in the 305‒335 nm range.86,87 Average stokes shifts measured for 2′-dG and dGMP are ~9632 and ~10092 cm-1 respectively. Thus, reorganization energy of dGMP estimated from the Stokes shift is ~ 5000 cm1

, which is higher than those estimated for any of the other purine bases. Several nucleobases,

such

as,

2'-deoxyadenosine-5'-monophosphate

(dAMP),

2-aminopurine

(2-AP),

2′-

deoxycytidine-5′-monophosphate (dCMP) and , 2'-deoxythymidine-5'-monophosphate (dTMP) have Stokes shift at 3070, 2950, 3040, 3500 cm-1 respectively. This large red shift derives from greater amount of stabilization of first singlet state of dGMP due to directional dipolar interaction with surrounding water molecules. 16 ACS Paragon Plus Environment

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The amplitude of the inertial component of solvation constituting the homogeneous part of the total linewidth broadening contribution was reported at 1050 and 1650 cm-1 following photoexcitation of 2′-dG in La and Lb state respectively.(see Table 2)25 We determine this component at 2550 cm-1 for GMP when excited within Bb absorption band. A significantly higher value of solvent reorganization energy is attributed to a larger change in dipole moment of guanine in the Bb state in comparison with that in La and Lb states with respect to the ground state. This interpretation is in agreement with ab initio computed dipole moment (10.17 Debye) of Bb state of Gua, which is 3 D more than that of ground state value, while dipole moments of La and Lb states do not differ by more than 1 D from that of the ground state.66 A higher transition dipole length (0.96 Å) of the Bb state compared to that of the Lb state (0.83 Å) is expected to polarize the charge distribution, leading to more interaction with polar solvent. The overall charge redistribution on the purine ring might play an important role in instantaneous alteration of local solvation structure. Exocyclic substitution can also affect the inertial component by modulating the ability of the base to interact with water molecule. Despite similar transition dipole strength compared to that of GMP, a twofold decrease in the magnitude of fastest component of water response is observed in 6-ClG.38 (See Table 2) The hydrophobic chlorine atom at 6th position in place of oxygen reduces the number of possible interactions with solvent molecules significantly. A similar effect is observed in case of introduction of exocyclic hydrophobic substituents such as methyl (5-methyluracil or thymine) or fluorine (5-fluorouracil) moiety to cause sharp reduction in the homogeneous linewidth component to 355 and 655 cm-1 respectively from that (1450 cm-1) obtained in uracil.88–90 The simulation also provides with a 21.2 fs solvent correlation time associated with 2550 cm-1 of reorganization energy. (Table 2) Following photoexcitation, such an ultrafast response of water has been reported from time dependent stokes shift,91,92 IR two-dimensional echo,93 threepulse photon echo,94 fluorescence up-conversion95 measurements on several chromophores. Presence of a sub-50 fs inertial response of the solvation shell was predicted from molecular dynamics simulations also.96–99 Using RR intensity analysis, a < 30 fs component of water dynamics for similar sized molecule, e.g., 6-ClG and tryptophan has been detected in previous reports from our lab. It has been reported that computed REPs are not responsive to change in Λ by ~50 cm-1.47,100 The widths rather than the intensities of REPs are sensitive to change in τ 17 ACS Paragon Plus Environment


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(=1/2πΛ). As the REPs reported in this study do not have experimental points on both sides of absorption maxima, it is difficult to put a certain limit on changes of this parameter. Presence of hydrophilic groups (-oxo and ribophosphate) on GMP can effectively increase the inhomogeneous linewidth (θ) in comparison to that of 6-ClG or 2′-dG. The presence of additional groups leads to many more allowed conformations and allied formation of slightly different micro-solvated environments of the solute. A consequence of this is ‘site broadening’ as each configuration has a slightly different zero-zero transition energy from the ensemble average. In GMP, there is an almost a fourfold increase of this component at 1350 cm-1 over the 350 cm-1 found for 6-ClG. The hydrophobic -chloro group in place of oxygen decreases overall interaction of 6-ClG with the surrounding solvent and potentially reduces the number of microsolvated clusters. 3.6 Caveats and future scope In our simulation we did not take into account a faint band observed at 679 cm-1 since the intensity could not be reliably measured at all excitation wavelengths. This band contributes to a maximum of 5% of the total integrated intensity of all RR active modes, and thus providing an upper-limit on the error due to its neglect. The robustness of the ∆s determined in the final fit can be seen in Fig 5 and 6 through the effect on the simulated absorption and RR cross-sections. We note that, the overall fit of REPs would be better constrained if experimental points on other side of the Bb absorption band are also provided. However, experimental RR spectra with excitations below 210 nm were not available due to limited laser wavelengths presently available. Even if RR intensities at higher energy excitation were available, we expect that interference from ther high lying electronic states will also contribute to the intensities. In that scenario, it would be more appropriate to use a more sophisticated two-state model, as employed for two lowest La and Lb excited states of 2ʹ-dG25 for the resonance Raman intensity analysis. Another important factor that is critical for determination of instantaneous distortions of different bonds of GMP are signs of the dimensionless ∆s. These are determined by gradients of potential energy along each normal coordinate in FC region of the Bb excited state. As in our previous report on 6-ClG38 we again found that, the signs of the gradients we obtained are the same irrespective of the choice of DFT functionals used. We note that the ground state normal mode vectors (matrix ‘Aji’ in Equation 4) that are necessary to project the dimensionless 18 ACS Paragon Plus Environment

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displacements onto changes in internal coordinates does vary with the number of explicit water molecules included in solvated model complex. Simulation of RR spectra of nucleobases on Bb and higher electronic states is not very common,101 as it is for the lower energy La and Lb electronic states.102–107 The uncertainties in signs of excited-state gradients obtained with TDDFT warrant more theoretical studies on higher-lying electronic states of GMP and of other nucleobases. Although challenging, measurement of Raman depolarization ratios across the 190 nm to 230 nm region would certainly reveal the nature of coupling of the close-lying electronic states. Highly desirable are further experimental measurements with sub-ps time-resolution, as well high-level calculations to understand the intricate couplings in these high energy ππ* electronic states. Conclusion We obtain good agreement between computed (9-meG•H2O) and experimental excitation energies of GMP by incorporating explicit water with guanine in the model complex. Experimentally measured resonance REPs of GMP within the Bb absorption band reveal that different vibrational degrees of freedom are responsible for coupling of high energy Bb to the lower energy Lb state and between the La and Lb states. Ring stretching (1580 and 1607 cm-1) and in-plane ring deformation (866 and 1020 cm-1) vibronically couple the Bb state with Lb state and assist internal conversion between them. Excitation to any of the three singlet states of Gua is not found to assist the formation of the guanine cation radical directly. Other than weakening of the C8‒N9 bond, photoexcitation to Bb state does not cause any distortion of the purine ring that lies along the photochemical reaction coordinate of various lesion formations. Bb excitation causes overall shrinkage of both the rings of Gua, in contrast to an expansion and contraction of pyrimidine and imidazole rings respectively in case of excitation to La or Lb states respectively. Large (72 %) contribution of the ultrafast inertial component of solvation towards total reorganization energy (3545 cm-1) of GMP is attributed to the presence of the hydrophilic ribophosphate group that takes part in directional dipolar interaction with surrounding water molecules. The presence of strong hydrogen bond accepting moieties on GMP results in a high value of inhomogeneous broadening linewidth of 1350 cm-1. These experimentally determined distortions would be of importance for assessing performance of different theoretical methods



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Tables Table 1. Vertical singlet excitation energies (∆Evert,PCM) in cm-1 (nm and eV in parenthesis), oscillator strengths (fPCM) and major orbital contribution to each transition of neutral GMP and comparison with published experimental and computed transition energies.

Computed Major orbital Contributio nb(%)

Type, Nomenc lature

State order

ππ*, La

S1

H→L (95)

ππ*, Lb

S2

H→L+1 (95)

nπ*, Snπ*

S4

H−2→L (81)

ππ*

S7

ππ*, Bb

S8

H−1→L (77) H→L+5 (9)

nπ*

S9

H−5→L (68)

ππ*

S10

H→L+6 (86)

ππ*

S11

H−3→L (91)

H→L+5(85)

∆Evert,PCMa,c cm-1 (nm/eV) 36300 (275.7/4.50) 40000 (250.1/4.96) 45300 (220.7/5.62) 47800 (209.1/5.93) 48200 (207.5/5.98) 48300 (206.9/5.99) 50500 (197.9/6.27) 50900 (196.4/6.31)

Experimental fPCMa,c

0.238 0.524 0.0005

Published ∆Evert

∆EExpc

∆EExpg cm-1 (nm/eV)

Extinction coefficient (ε)c (L mol-1 cm-1)

(eV)

cm-1 (nm/eV)

4.65d, 4.73e, 4.46f 5.10d, 5.11e, 4.71f 5.53d, 5.98e, 5.64f

36297 (277.4/4.47) 40322 (248.0/5.00) 45005 (222.2/5.58)

36297 (275.5/4.50) 40322 (248.0/5.00) 45977 (217.5/5.70)

12083 12080h

6.49e, 6.23f

50075 (199.7/6.21)

50000 (200.0/6.20)

17250

(187.0/6.63)

187.8 (6.60)

7063.8

0.080 0.348 0.038 0.006 0.166

6.59e 6.72e, 6.53f

a

computed on 9-meG•6H2O complex at TD-B3LYP method with 6-311+G(2d,p) basis set and clustercontinuum model as described in computational method. Six explicit water molecules from first solvation shell are included and non‒equilibrium solvation using self consistent reaction field (SCRF-PCM or PCM) model using water as solvent is employed; bPercentages are calculated as 100 x twice the squares of the coefficients in the CI expansion of TD-DFT wave functions; cthis work; dRef 72, computed with TD-X3LYP method using cluster-continuum solvation approach; eRef 66, in gas phase with CASSCF method on N9H-Gua; fRef 67, in gas phase with real time TD-DFT implemented in time domain and on N9H-Gua; gcollected from Ref 66, Average experimental λmax determined from UV-Vis absorption in liquid, linear dichroism, circular dichroism, magnetic circular dichroism and polarized absorption spectroscopy in crystalline state of guanosine, guanine and 9-ethylguanine. hRef 56.


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Table 2. Parameters used in self-consistent simulation of REPs and absorption spectra Molecule 6-ClGa 2′-dGb GMP

Excited State Bb La Lb Bb

E0(cm-1)

Λ(cm-1)

τ(fs)

θ(cm-1)

M(Å)

λint(cm-1)

λS(cm-1)

λ(cm-1)

Best fit Best fit

45700 35750

350 65.93

15.2 80.53

360 900

1.11 0.56

112 153

1220 1050

1332 1203

Best fit Best fit Set 1 Set 2

37600 49100 48600 49360

82.73 250 320 220

64.23 21.2 16.6 24.1

900 1350 1020 1680

0.83 0.96 0.96 0.96

904 995 1433 637

1650 2550 3250 2200

2554 3545 4683 2837

a

Ref 38; bRef 25. Instead of dimensionless displacement ∆, the slopes (β/ħ) along normal modes of vibrations in dimensionless coordinate are provided for 2′-dG. For a direct comparison with parameters associated with Bb electronic state these slopes are converted to ∆s via the equation, ∆k= ‒(βk/ωk). Mode specific reorganization energy (λk) is computed using the relation, λk=(∆k2ħωk)/2. ħ is unity in atomic unit. Total internal reorganization energy is computed by summing overall such λk’s. Inverse of solvent relaxation time (Λ) is calculated from the relation between solvent reorganization energy (homogeneous linewidth) and coupling strength (D) between solvent coordinate and electronic transition, D2 = 2λskBT and the assumption of Λ/D=0.1 in high temperature limit. Then solvent correlation time τ is estimated from the relation, Λ = 1/2πcτ where c is the speed of light. Set 1 and Set 2 were obtained with two sets of deltas obtained by decreasing and increasing 20% those values (see Table 4) in the best fit.



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Table 3. Internal coordinates and displacements for the modes that undergo largest distortion following photoexcitation of GMPa in La, Lb and Bb state. Internal coordinates description Bond length N1–C2 C2=N3 C2–N2 N3–C4 C4=C5 C5–C6 C6–N1 C5–N7 N7=C8 C8–N9 N9–C4 C6=O N9– Cmethyl a

Value in ground state (Å) 1.378 1.330 1.339 1.349 1.392 1.418 1.397 1.384 1.307 1.376 1.368 1.245 1.457

Changes in La state (Å) 0.00 -0.00 0.01 0.02 -0.01 -0.04 0.01 0.01 -0.03 0.02 -0.04 -0.02 -0.02

Changes in Lb state (Å) 0.00 0.03 0.06 0.11 -0.02 -0.05 0.07 0.07 -0.07 0.01 -0.01 -0.03 -0.06

Changes in Bb state (Å) -0.09 -0.02 -0.00 0.05 -0.04 -0.03 -0.09 0.00 -0.01 0.07 -0.07 -0.04 -0.05

GMP in solution is modeled as 9-meG•6H2O.


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Table 4. Experimental and computed wavenumbers, PEDs with dimensionless displacements and estimated internal reorganization energies of most important five RR bands of GMPc. Experimental (cm-1)

Computed wavenumbersa (cm-1)

1076

1088

1177

1132

1365

1377

1580

1622

1607

1665

PED (%)

b

‒ str N1C2 (11) + be C2N3C4 (15) ‒ C5N7C8 (12) ‒ str N3C2 (20) + N1C2 (10) + be N2H2bO (22) str C8N9 (12) + be C5N7C8 (16) str N3C2 (16) – N2C2 (13) – be H1N1C2 (27) – N1C2N3 (10) str C6O6 (15) – be H1N1C2 (15)

|∆|c (Dimensionless)

Mode specific internal reorganization energy, λk (cm-1)

0.43

99.5

0.43

108.8

0.68

315.6

0.34

91.3

0.40

128.6

a

B3LYP/6-311+G(2d,p)//PCM and not scaled; busing VEDA 4.0, sign indicates relative phase of movement of internal coordinates of atoms; cobtained form best fit parameters in Table 2; str: stretching; be: bending; cGMP in solution is modeled as 9-meG•6H2O. These five RR modes contribute ~ 75 % of total internal reorganization energy. See table S6 in Supporting Information for complete assignment.



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Page 32 of 38

Figures

Figure 1. Structure of neutral aqueous GMP obtained by energy minimization at B3LYP/6311+G(2d,p)//PCM level of theory. Aqueous GMP was modeled as 9-meG•6H2O complex as shown above. All H-bond distances (in Å) between water molecules and the base along are labeled. Conventional numbering of ring atoms is indicated. Directions of computed transition dipole moments hi) for Bb and S11 states are shown with bold arrows. Indicated magnitude of the transition dipole moment (g vectors is magnified by a factor of two (for Bb) and three (for S11) over the calculated value for improved hhhhhhhhhhhhhhhhi visualization. Computed hgi of Bb and S11 state are oriented at 86.1º and 8.4º with respect to j k − lm and have a magnitude of 2.38 and 1.07 Au respectively. Indicated magnitude of the transition dipole moment vectors is magnified by a factor of 1.75 over the calculated value for improved visualization.


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Figure 2. (a) Computed molecular orbitals of GMP involved in the electronic transition observed in experiments at 210 nm; (b) bottom: deconvolved experimental spectra with standard spectroscopic labels of the bands and λmax; top: computed absorption spectra, excitations, and nature of the lower and higher excited states of GMP. The electronic absorption spectrum was computed using TD-B3LYP/6311+G(2d,p)//PCM method on 9-meG•6H2O complex. Computed band positions are depicted with a Gaussian line shape of fixed line width (3500 cm-1). H and L stand for HOMO and LUMO orbitals respectively. Absorption cross-section was determined following the procedure described in the experimental method section. Computed states of GMP are described in table 1. Region of absorption spectra (210 – 230 nm) covered in measuring REPs of resonant modes of GMP is marked with arrows in the bottom panel of b.



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Page 34 of 38

Figure 3. UVRR spectrum of GMP (1 mM, pH 6.8 in milliQ water) obtained with excitation wavelength of 212 nm with incident laser power of 0.6 mW. The band at 932 cm-1 corresponds to sodium perchlorate that was used as internal intensity standard. Normal mode descriptions of the most important RR

bands of GMP (see Table 4) energy are also labelled. Abbreviations; str., bond stretching; rock, rocking motion; be., bending; breath., in-plane ring breathing, sciss., scissoring motion.


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Figure 4. Resonance Raman spectra of GMP (1 mM, pH 6.8 in milliQ water) at eight different excitation wavelengths in aqueous solution. Spectra show variation in the Raman intensity of each band as the excitation wavelength is tuned across the electronic absorption band (Fig. 2, panel b, bottom). Spectra are normalized to the intensity of the 932 cm-1 band of sodium perchlorate used as internal intensity standard (asterisk). Positions of five most important RR bands (described in Figure 3 and Table 4) of GMP are also labelled.



4.5

4.8

0.75 a

5.1 5.4 0.08 b Absorption 7 x 10

-1

0.08

866 cm

c

1046 cm

f

1210 cm

i

1413 cm

-1

0.50 0.04

0.04

0.25 0.00

d

0.00

-1

1076 cm

e

-1

1177 cm

0.2

0.12

2

-7

Cross-section (Å / molecule x 10 )

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

Page 36 of 38

0.00 g 0.06

-1

1255 cm

0.0

-1

0.04

0.1

0.06

0.00

0.02

-1

h

1365 cm

0.4

0.00

-1

0.04

0.04 0.2

0.02

0.02 0.00

4.3

4.4

4.5

4.6

4.7

0.0

4.3

4.4

4.5

4.6

4.7

-1

0.00

4.3

4.4

4.5

4.6

4.7

4

Wavenumber (cm x 10 )

Figure 5. Experimental and simulated Raman excitation profiles and absorption cross-sections of GMP in the 210-230 nm region. (a) Experimental (dashed) and simulated (solid) absorption spectrum. (b–i) Experimental (points) and simulated (solid) Raman excitation profiles for eight resonant modes of GMP. The simulated absorption and Raman excitation profiles were obtained by using the parameters in Table 2.


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Figure 6. Experimental and simulated Raman excitation profiles of the other four resonant modes of GMP. The simulated Raman excitation profiles were obtained by using the same sets of parameters as in Figure 5.



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Page 38 of 38

Figure for Table of Content (TOC) entry

Change (in Å) in nuclear coordinates of GMP in Lb (in red) and Bb (in blue) excited states.


Ultrafast Nuclear Dynamics of Photoexcited Guanosine-5

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