Excited-State Dynamics in 6-Thioguanosine from the Femtosecond to Microsecond Time Scale

Published: March 08, 2011

r 2011 American Chemical Society 3263 dx.doi.org/10.1021/jp112018u | J. Phys. Chem. B 2011, 115, 3263–3270

ARTICLE

pubs.acs.org/JPCB

Excited-State Dynamics in 6-Thioguanosine from the Femtosecond toMicrosecond Time ScaleChristian Reichardt, Cao Guo, and Carlos E. Crespo-Hern�andez*

Department of Chemistry and the Center for Chemical Dynamics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland,Ohio 44106, United States

1. INTRODUCTION

6-Thioguanine (6tGua) and other purine derivatives are mem-bers of a family of biomolecules known as pro-drugs, which arewidely prescribed for maintenance therapy of acute lymphoblas-tic leukemia, for inflammatory bowel disease that is unresponsiveto steroids,1 and for gliomas.2,3 In particular, 6tGua is a cytotoxicagent of clinical relevance4-6 and has been used in cross-linkingstudies and as a site-specific optical probe.7,8

6-Thioguanosine (6tGuo), a metabolite of 6tGua,9,10 is asingle-atom-substituted guanine base analogue that absorbs inthe ultraviolet A region (UVA) of the electromagnetic spectrum(Figure 1). Previous works have shown that irradiation of DNAcontaining 6tGuo with UVA light can damage DNA causing celldeath.11-14 Furthermore, the DNA of patients treated withimmunosuppressant and anticancer drugs azathioprine or mer-captopurine incorporates 6tGuo after metabolization.9,10 Theskin of these patients is sensitive to UVA radiation, and long-termtreatment can result in extremely high incidences of sunlight-induced skin cancer.15 Studies have shown that radiation of6tGuo with UVA light results in the formation of reactive oxygenspecies in cells treated with thiopurines.16-18 It is expected thatthe triplet excited state of 6tGuo plays a major role in itsphototoxicity, but direct evidence of the population of the tripletstate after UVA light absorption and its quantum yield is lacking.

In this contribution, we investigated the excited-state dy-namics of 6tGuo in phosphate buffer and in acetonitrile solu-tions. As recently shown for the DNA thymidine analogue,4-thiothymidine (4tThd),19,20 replacement of the oxygen inthe carbonyl group of guanosine by a sulfur atom results in the

ultrafast population of the triplet state in high yield due toenhanced spin-orbit and vibronic coupling between the singletand triplet manifold. A triplet yield of 0.8 ( 0.2 is estimated inaqueous buffer solution with reference to that of 4tThd.19 Ourresults support the idea that when 6tGuo is incorporated in DNAit can act as an efficient UVA photosensitizer causing oxidativeDNA damage and cell death. Finally, the steady-state photo-physics and excited-state dynamics of 6tGuo are compared withthose of the DNA/RNA guanine monomers.

2. EXPERIMENTAL METHODS

2.1. Chemicals and Steady-State Measurements. 2-Amino-6-mercapto-9-(β-D-ribofuranosyl)purine hydrate (>98% purity),also known as 6-thioguanosine or 6-mercaptoguanosine, wasobtained from Carbosynth Limited, Berkshire, UK, and used asreceived. Phosphate buffer solutions were freshly prepared using0.24 g of sodium dihydrogen phosphate and 0.177 g of disodiumhydrogen phosphate dissolved in 200 mL of ultrapure water andadjusted to pH 7.0 using a diluted solution of NaOH. The steady-state absorption spectrum was measured at room temperatureusing a Cary 100 spectrophotometer (Varian, Inc.). Fluorescencemeasurements were recorded using a Cary Eclipse spectrofluori-meter (Varian, Inc.). The absorbance of the solutions at theexcitation wavelength of 310 nm was <0.2 absorbance units.

Received: December 17, 2010Revised: February 16, 2011

ABSTRACT: Patients treated with the immunosuppressantand anticancer drugs 6-thioguanine, azathioprine, or mercapto-purine can metabolize and incorporate them in DNA as6-thioguanosine. The skin of these patients is sensitive toUVA radiation, and long-term treatment can result in extremelyhigh incidence of sunlight-induced skin cancer. In this contribu-tion the photophysics of 6-thioguanosine have been studied inaqueous buffer solution and in acetonitrile after excitation withUVA light to provide mechanistic insights about the origin of itsphototoxicity. It is shown that most of the initial excited-state population in the S2(ππ*, La) state decays by ultrafast intersystemcrossing to the triplet manifold. A triplet quantum yield of 0.8( 0.2 is determined in aqueous buffer solution. Aminor fraction of theS2 population bifurcates on an ultrafast time scale to populate the S1(nSπ*) state, which decays back to the ground state in tens ofpicoseconds. Quantum-chemical calculations that include solvent effects support the experimental results. The high triplet yield of6-thioguanosine, which we argue can result in photosensitization of molecular oxygen and photooxidative DNA damage, is proposedto explain the high phototoxicity exhibited by these pro-drugs in patients upon sunlight exposure. Finally, the experimental andcomputational results for 6-thioguanosine are compared with those reported for the DNA/RNA guanine monomers.

3264 dx.doi.org/10.1021/jp112018u |J. Phys. Chem. B 2011, 115, 3263–3270

The Journal of Physical Chemistry B ARTICLE

2.2. Femtosecond Broadband Transient Absorption Spec-troscopy. The femtosecond broadband transient absorptionspectrometer and data analysis procedure have been describedin detail elsewhere.19,21 The instrument setup conditions wereanalogous to those reported recently for the investigation of4-thiotymidine.19,20 Briefly, a Quantronix Integra-i/e 3.5 Lasergenerating 100 fs pulses at 800 nmwith a repetition rate of 1 kHz isused to pump an optical parametric amplifier. The OPA output istuned to the excitationwavelength of 340 nm, and the pulse energyis attenuated to 1 μJ at the sample position. The setup contains anoptical delay line that provides amaximum time delay of 3.2 ns andan all-reflective optics white light continuum generation schemefor broadband probing. A CaF2 crystal of 2mm thickness was usedfor continuum generation giving access to the spectral range from330 to 670 nm when focusing a small fraction of the 800 nm pulseinto the crystal. The broadband probe pulses were split into twofractions, recollimated, and focused into optical fibers leading intothe spectrometer/CMOS detection units. One of the two beamspasses a 2 mm optical path length sample cell for probing thesignal, while the other beam is used as reference. For thegeneration of a reference signal, every other pump pulse is blockedby a synchronized chopper wheel, thus generating an alternatingsequence of spectra with and without sample excitation.Data acquisition and data analysis are performed using a home-

made LabView software (National Instruments, Inc.) and a globalfitting subroutine set up in the Igor Pro 6.12A software(Wavemetrics, Inc.), respectively.21 The instrument responsefunctionwas approximately 200 fs as determined from the coherentsignal seen in solvent-only scans. For the global fitting analysis, 24representative kinetic traceswere selected for each data set coveringthe full range of probe wavelengths (330-670 nm). The kinetictraces were analyzed by globally fitting to a sum of two exponentialfunctions plus a constant offset, convoluted with a Gaussianresponse function with full-width-at-half-maximum of 200 fs torepresent the instrument response function. Four independentdata sets (i.e., data recorded on different days) were used in theanalysis. The time constants and time zero were linked for all thetraces, while the amplitudes were left wavelength-dependent. Allreported uncertainties are twice the standard deviation (2σ).The absorbance of the 6tGuo was typically in the range of 0.75

to 1.0 in buffer solutions and in the range of 0.20 to 0.24 inacetonitrile at the excitationwavelength using a 2mmoptical pathlength cell. This corresponds to 6tGuo concentrations of ca.0.2 mM in aqueous buffer solutions and of 0.05 mM in acetoni-trile. The sample in the probed volumewas continuously renewedby a magnetic stirrer to avoid re-excitation of the excited volumeby successive laser pulses using a Teflon-coated stir bar. Solutionswere carefully monitored for photodegradation using a spectro-meter, and the samples were replaced with fresh solutions asneeded. Importantly, no changes in the transient absorptionspectra or decay signals of 6tGuo were observed in solutions thatshowed ∼10% or less decrease in ground-state absorption at∼340 nm after the time-resolved experiments were completed.Transient absorption spectra of 6tGuo were recorded on the

picosecond tomicrosecond time scale using a photonic crystal fiberfor probe light generation in the spectral range from ∼375 to675 nm where the pump-probe delay is electronically controlled(Eos,Ultrafast Systems, LLC).TheEos light source is convenientlycoupled to our femtosecond transient absorption spectrometer,21

which allows us to use identical experimental conditions anddetection spectrometer. Data acquisition and analysis using theEos light source have been explained in detail previously.21

2.3. Estimation of the Triplet Extinction Coefficients andYield.We estimated the extinction coefficients of the triplet stateof 6tGuo in buffer solution using the singlet depletion method.22

In using this method, we assume that the absorbance of anyexcited-state species does not change significantly over thespectral region where the ground-state bleaching is observed atthe time delay used to do the measurement (Δt = 2.5 ns).23 Wehave examined the accuracy of a singlet depletion method usinga thymidine analogue, 4-thiothymidine (4tThd), for which theextinction coefficient of the triplet state has been previouslyreported to be 2800 ( 700 M-1 cm-1 at 520 nm in acetonitrileby Harada et al.24We estimated an extinction coefficient of 2600( 500 M-1 cm-1 for 4tThd in acetonitrile using this method,which agrees with the reported value at the same probewavelength.24 Hence, we determined a triplet extinction coeffi-cient of 3400( 700 M-1 cm-1 at 520 nm for 6tGuo in aqueousbuffer solution using the singlet depletion method, while anextinction coefficient of 1800 ( 400 M-1 cm-1 was measuredfor 4tThd in aqueous buffer solution.Once the triplet state extinction coefficients of 6tGuo and 4tThd

were obtained in aqueous buffer solutions, we used a comparativemethod to estimate the triplet yield of 6tGuowith reference to thatreported for 4tThd under similar conditions.19,24 Back-to-backtransient absorption spectrawere recorded for 6tGuo and 4tThd inaqueous buffer solution, both solutions having an optical densityof 1.0 at the excitation wavelength. The relative actinometrymethod22 was then used to determine the triplet yield of 6tGuo.2.4. Quantum Chemical Calculations. Quantum chemical

calculations were performed to assist in the interpretation of theexperimental data. Ground- and excited-state calculations wereperformed using the Gaussian 03 suite of programs,25 as de-scribed previously.19-21 Briefly, the ground-state geometry of6tGuo was optimized at the density functional level of theory(DFT) by using the B3LYP functional26-28 and the 6-311þþG-(d,p) basis set, while vertical excitation energies were estimatedby using the time-dependent implementation of DFT with theparameter-free PBE0 functional29 and the 6-311þþG(d,p) basisset. Herein we have focused on the anti C20-endo (North) sugarconformation, which is the favored conformation in B-formDNA.30 The optimized structures were verified as true localminima on the ground-state potential energy surface by establish-ing that the Hessian matrix did not exhibit imaginary eigenvalues.Solvent effects on the ground-state geometries and verticalexcited-state energies of 6tGuo were modeled using the polariz-able continuum model (PCM)31 with the integral equation

Figure 1. Absorption spectra of 6tGuo in phosphate buffer pH 7.0 andacetonitrile. Inset: ground-state optimized molecular structure of theanti C20-endo 6-thioguanosine (6tGuo) in water at the B3LYP/IEFPCM/6-311þþG(d,p) level of theory.



formalism (IEFPCM).32 The equilibrium solvation limit is used,where solvent electrons and nuclear polarizations equilibratewith the molecular excited state.33

3. RESULTS AND DISCUSSION

3.1. Steady State Absorption and Emission Spectra of6tGuo in Aqueous Buffer Solutions and in Acetonitrile. Theabsorption spectra of 6tGuo in aqueous buffer solution and inacetonitrile are shown in Figure 1. The lowest-energy absorptionband has a maximum at (342 ( 1) nm and an extinctioncoefficient of (2.3( 0.1)� 104M-1 cm-1 at the samewavelengthin aqueous buffer solution, while it has a maximum and extinctioncoefficient in acetonitrile at (346( 1) nm and (2.2( 0.2)� 104

M-1 cm-1, respectively. Higher electronic states in the UVCregion are more perturbed by solvation than the electronic statesin the UVB and UVA spectral region. The bathochromic shift ofthe UVA band(s) on going from aqueous buffer solution toacetonitrile can be explained by the higher electron-pair donatingability of acetonitrile versus water.34 The steady-state absorptiondata are in good agreement with the vertical excitation energiesand oscillator strengths reported in Table 1.Attempts were made to record the emission spectra of 6tGuo

in aqueous buffer solution and in acetonitrile at room tempera-ture. However, no emission was detected within the sensitivity ofthe spectrometer used. Interestingly, fluorescence emission withan excitation spectrum corresponding to a photoproduct wasobserved after considerable irradiation of 6tGuo aqueous solu-tions, which strongly suggests the formation of a relativelyfluorescent photoproduct (data not shown). Hence, precautionswere taken when measuring the transient absorption datareported in this work to avoid contamination of the transientsignal by signals originating from photoproduct formation. Thelack of observable steady-state fluorescence in 6tGuo is sup-ported by the quantum chemical calculations presented next.3.2. Electronic Structure Calculations. Ground-state opti-

mizations of 6tGuo were performed in vacuum, acetonitrile, andwater at the B3LYP/IEFPCM/6-311þþG(d,p) level of theory.Figure 1 shows the optimized geometry in aqueous solution.Similar geometries were obtained in vacuum and in acetonitrile(not shown). The optimized geometries were then used toestimate the vertical excitation energies in the correspondingsolvent environment. Our calculations show that a minimum offive excited electronic states must be considered in describing thephotochemistry of 6tGuo in the UVA region of the electromagnetic

spectrum. Table 1 shows the vertical excitation energies for the twolowest singlet excited states and for the three lowest triplet states of6tGuo in vacuum, acetonitrile, and water as determined at the TD-PBE0/IEFPCM/6-311þþG(d,p) level of theory. The character ofthe excited states was estimated from the effect that the solvent hason the excitation energies and from inspection of the molecularorbital transitions describing each excited state. Our results for6tGuo in vacuum are in very good agreement with those presentedpreviously for 6tGua at theCNDO/S andTD-B3LYP/6-311þþG-(d,p) levels of theory.35-37However, our calculations are apparentlythe first ones performed for 6tGuo in vacuum and in solution.The results shown in Table 1 suggest that the lower-energy

singlet excited state (S1) of 6tGuo has nSπ* character and negli-gible oscillator strength, while the second excited singlet state(S2, La) has ππ* character and a strong oscillator strength. Forthe latter we use the nomenclature of Platt-Murrell,38,39 wherethe La label represents the state with the highest contribution ofthe LUMO r HOMO single particle transition. In addition,there are three triplet states lower in energy than the S2 state. Theenergy gap between the S2 and the T3 state is smaller than 0.1 eVin the Franck-Condon region in acetonitrile and aqueoussolutions, which suggests that these two states can have strongspin-orbit and vibronic coupling in the Franck-Condon regionof the potential energy surface. Hence, excitation of 6tGuoat 340 nm is predicted to initially populate the S2(ππ*) state,

Table 1. Vertical Excitation Energies for antiC20-endo 6tGuoat the TD-PBE0/IEFPCM/6-311þþG(d,p) Level of Theorya

state vacuum/eVa ACN/eVb H2O/eVc

T1(ππ*) 2.58 2.74 2.74

T2(nπ*) 2.91 3.39 3.42

S1(nSπ*) 3.23 (0.00) 3.62 (0.00) 3.64 (0.00)

T3(nπ*) 3.52 3.87 3.88

S2(ππ*, La) 4.07 (0.28) 3.96 (0.45) 3.96 (0.45)

ΔE(S2 - T3) 0.55 0.09 0.08

ΔE(S1 - T1) 0.65 0.88 0.90aGround-state optimizations were performed at the B3LYP/IEFPCM/6-311þþG(d,p) level of theory in vacuum, acetonitrile, and water,respectively. Oscillator strengths for the singlet state are shown inparentheses.

Figure 2. Transient absorption spectra of 6tGuo in aqueous buffersolution at pH 7.0 after 340 nm excitation (gray vertical bar): (a) earlytime delays and (b) time delays from 1.8 to 540 ps. The stimulatedRaman emission band of water at 385 nm is observed at short timedelays.



followed by internal conversion to the S1(nπ*) state and/ordirect intersystem crossing (ISC) to the T3(nπ*) on an ultrafasttime scale in both solvents.If ISC occurs directly from the S2(ππ*) state, the receiver

T3(nπ*) state is expected to internally convert to the T1(ππ*)state on an ultrafast time scale. If the S1(nπ*) state is populatedby ultrafast internal conversion, then ISC to the T1(ππ*) statecan still be favorable, as predicted by the El-Sayed propensityrules.40,41 The S1(nπ*) can also decay back to the ground statenonradiatively. Yet another possibility is ultrafast branching ofthe initially excited S2(ππ*) state to populate both the S1(nπ*)and T3(nπ*) states. Of course, ISC from the S2(ππ*) tothe T2(nπ*) cannot be ruled out on the basis of these verticalcalculations, while ISC from S1(nπ*) to T2(nπ*) is unlikely. Ineither case, the calculations suggest that fluorescence emissionfrom 6tGuo should be negligibly small in solution because of thepotential efficient participation of one or more of these non-radiative decay pathways. This prediction is in agreement withthe experimental observations reported in this work.3.3. Transient Absorption Experiments of 6tGuo in Solu-

tion. Figure 2 shows the transient absorption spectra of 6tGuo inaqueous buffer solution exciting at 340 nm. A negative absorp-tion band is observed below∼350 nm, while positive absorptionbands with maxima around 375 and 600 nm are observed at earlytime delays. The negative absorption band with maximumamplitude at ∼340 nm is assigned to ground-state depopulation

(bleaching) of 6tGuo in agreement with the steady-state absorp-tion spectrum (Figure 1). The dynamics of the positive transientabsorption bands with maxima at 380 and 620 nm at delay timeslonger than ∼2 ps are identical, suggesting that they should beassigned to a single excited-state species. Initially, an increase inexcited-state absorption is observed above 350 nm with a simult-aneous partial repopulation of the ground state as shown bythe bleaching signals below 350 nm. In fact, the kinetics of thebleaching signal and of the positive absorption bands can besatisfactorily fitted globally to a sum of two exponential functionsplus a time-independent constant. Similar transient absorptionsignals are observed in acetonitrile (Figure 3).Figure 4 shows representative decay signals and best-fit results

for 6tGuo in aqueous buffer solution and in acetonitrile. Theassociated globally fitted lifetimes are (0.31( 0.05) ps and (80(15) ps in aqueous solution and (0.36( 0.04) ps and (32( 5) psin acetonitrile. Analogous to the excited-state dynamics of4tThd,19,20 the first lifetime is assigned to the ultrafast populationof the triplet state in 6tGuo. We argue that ISC to the tripletmanifold occurs directly from the S2(ππ*, La) state to theT3(nSπ*) state based on the calculated vertical excitation en-ergies, which predict an energy gap of less than 0.1 eV betweenthese two states (Table 1). Note that the energy gap between the

Figure 3. Transient absorption spectra of 6tGuo in acetonitrile after340 nm excitation: (a) early time delays and (b) time delays from 1.5 to280 ps. The stimulated Raman emission band of the solvent at∼380 nmis observed at short time delays.

Figure 4. Representative decay signals in (a) aqueous buffer solution atpH 7.0 and (b) acetonitrile at the reported probe wavelengths. Bestglobal-fit curves are shown by solid lines.



S1(nSπ*) and the T1(ππ*) state is close to 1 eV in solution(Table 1), which suggests that ISC between these two states inthe subpicosecond time scale is less likely to occur as a majordeactivation channel. In addition, even though the singlet-triplet energy gap between the S1 and T2 states is smaller thanthat between the S1 and T1 states, the propensity rules40,41

predict that ISC from S1(nπ*) to T2(nπ*) should be lessfavorable, as noted in the previous section. Clearly, ab initioquantum dynamic calculations are needed to provide a more in-depth mechanistic model of the singlet and triplet excited statesparticipating in the electronic relaxation pathways in 6tGuo.Further evidence that the long-lived absorption bands ob-

served above 350 nm should be assigned to the triplet state wasobtained by monitoring the decay of the transient absorptionspectra to the microsecond time window in N2- and air-saturatedconditions. The transient absorption bands decay monotonicallyin the spectral region from 400 to 670 nm showing that they aredue to a single species (Figure 5a). Figure 5b shows that thedecay of the long-lived species can be slowed down by removingmolecular oxygen from the solution. A decay lifetime of (720 (10) ns is obtained in N2-saturated conditions, while (460 ( 15)ns is measured in air-saturated conditions. The 1.6-fold increasein lifetime in N2-saturated conditions shows that molecularoxygen can quench the long-lived species, indicating that itshould be assigned to the triplet state.We have estimated the triplet yield of 6tGuo in aqueous buffer

solution using 4tThd as a reference standard (see ExperimentalMethods).19,24 A triplet yield of 0.8( 0.2 was obtained. The hightriplet yield of 6tGuo together with the observed quenching ofthe triplet state by O2 are likely to result in high yield of singlet

oxygen (1O2), which is known to be effective in oxidizingDNA.42

These results can explain previous observations that irradiationof DNA containing 6tGuo with UVA light damages DNA andresults in cell death.11-14

The assignment of the second lifetime is less clear due to itslow amplitude and the possible overlap of transient absorptionbands during the time scale that represent this dynamical process.A close inspection of the early dynamics in Figures 2 and 3 revealsthat the transient absorption spectra do not show evidence of therelatively strong UV band with a maximum around 370 nm attimes shorter than∼0.3 ps. In addition, the ground-state bleachingsignal reaches a maximum approximately at a time delay of∼0.3 ps,followed by a partial recovery on the time scale of tens of picose-conds (τ2) (Figure 4). These observations suggest that there isanother minor relaxation pathway with ∼15% relative amplitudeoverlapping with the initial ultrafast population of the triplet state.This minor pathway decays back to the ground state on a time scalerepresented by the second lifetime.We propose that the minor relaxation pathway observed in the

bleaching and visible excited absorption band is due to the ultra-fast population of the S1(nSπ*) state (i.e., ultrafast internalconversion from the S2(ππ*, La) state), which decays back tothe ground state in tens of picoseconds in both solvents. We notethat S0 r S1(nOπ*) internal conversion in the pyrimidine basesalso occurs on a picosecond time scale.43 A mechanism in whichthe initial wave packet populates both states simultaneously withthe major fraction of the population going to the S2(ππ*, La)state and a small fraction populating the S1(nSπ*) state cannot beruled out unequivocally.33 This difficulty is due to the overlap ofthe transient absorption bands (Figures 2 and 3) and to the closeproximity of the S2(ππ*, La) and S1(nSπ*) states in the Franck-Condon region of 6tGuo. We favor the ultrafast internal con-version pathway because the oscillator strength of the forbiddenS1(nSπ*) vertical transition is close to zero at the TD-PBE0 levelof theory (Table 1).The partial increase in the transient absorption band with

maximum around 370 nm is tentatively assigned to solvation dyna-mics in the triplet manifold occurring in the same time scale as S1 toS0 internal conversion. It is conceivable that the reorientation of thesolvent molecules in response to the change in the dipole moment

Figure 5. (a) Transient absorption spectra of 6tGuo in aqueous buffersolution at pH 7.0 in air-saturated conditions from 8 ns to 5.5 μs timedelay. (b) Decay absorption signals (open circles) in aqueous buffersolution at pH 7.0 at the specified experimental conditions. Best global-fit curves are shown by solid lines.

Scheme 1. Proposed Kinetic Modela

a Excitation of 6tGuo at 340 nm in solution results in ultrafast bifurcationof the excited-state population in the S2 to populate the triplet state(major channel) or to internally convert to the S1 state (minor channel).The nonradiative decay pathways are characterized by τ1∼ 0.3 ps in ourtransient absorption experiments. Intersystem crossing to the tripletmanifold results in population of the lowest-energy triplet state with∼0.8 quantum yield. The triplet state decays to the ground state with alifetime of hundreds of nanoseconds (τ3). The S1 state decays to theground state in tens of picoseconds (τ2).



and nuclear coordinates of 6tGuo in the T1 state relative to the S2state can slightly increase the Tn r T1 absorption cross sectionsof the UV band. A kinetic model that summarizes our interpret-ation of the experimental and computational results is presented inScheme 1.3.4. Comparison of the Excited-State Dynamics in 6tGuo

with those in 9N-Substituted Guanine Monomers. In thissection we comment on the excited-state dynamics in 6tGuo ascompared to those in the DNA/RNA guanine monomers.Previous experimental44-53 and computational works54-63 haveshown that the interpretation of the time-resolved photochem-istry of the guanine monomers is intricate. This is largely due tothe close proximity of several electronic states, to the presenceof multiple conical intersections (CIs) between the potentialenergy hypersurfaces, to the presence of multiple low-energytautomers, and to the sensitivity of the energy of these electronicstates to the microenvironment surrounding the guanine chro-mophore. The experimental and computational results presentedabove show that 6tGuo is not an exception. However, the knowl-edge gathered in recent years on the excited-state dynamics in theguanine monomers can provide some insights about the deactiva-tion mechanism in 6tGuo. In the following we focus on thebiologically relevant 9N-substituted guanine monomers becausetheir photophysics can be compared directly with the 9N-ribonu-cleoside studied in this work.Early transient absorption and up-conversion experiments

have shown that UV excitation at 267 nm of the guanine mono-mers in solution results in efficient and ultrafast decay of theS1(ππ*) state population to the ground state.44,48,49,64 Morerecently it has been shown that the excited-state dynamics in theRNA33 and DNA65 guanosine 50-monophosphate in solution(GMP and dGMP hereafter) are more precisely described by abiexponential decaymechanismwhere the relaxation pathways ofthe S1(ππ*) state population occur from two different regions ofthe potential energy surface. The experimental measurements forGMP were complemented with vertical and optimized excited-state calculations on 9-methylguanine (9MG) at the TD-PE0level of theory that include solvent effects.33 Karunakaran et al.33

proposed that excitation at 267 nm results in simultaneouspopulation of the S2(ππ*, Lb) and the S1(ππ*, La) states. TheS2(ππ*, Lb) state internally converts to the S1(ππ*, La) state in<100 fs time scale,33 while the population in the S1(ππ*, La) stateinitially stays near the Franck-Condon region where the poten-tial energy surface is almost flat and the purine ring has a planargeometry (τ ∼ 0.2 ps). The population then evolves toward adifferent region in the potential energy surface where the guaninechromophore attains a nonplanar geometry that connects theS1(ππ*, La) state with the S0 state through a CI (τ ∼ 0.9 ps).33

This CI is reached by pyramidalization at C2 and out-of-planemotion of the amino group as has been documented previouslyin the gas phase (vacuum) at various levels of theory.54,56,57,59,60

Furthermore, the involvement of nπ* and πσ* states in theexcited-state dynamics of GMP in water was ruled out on thebasis of the transient absorption results and quantum-chemicalcalculations.33

The kinetic model presented by Karunakaran et al.33 for GMPis in good agreement with the interpretation of the steady-stateand up-conversion fluorescence experiments by Gustavsson andco-workers on dGMP in aqueous solutions.65 The authors observedthat the initial fluorescence anisotropy was smaller than 0.2 at allwavelengths investigated and that the fluorescence shows significantinhomogeneity in both steady-state and up-conversion experiments.

These results were taken as evidence that excitation at 267 nmresults in approximately equal population of the S2(ππ*, Lb) and theS1(ππ*, La) states, where the initial population in the S2(ππ*, Lb)state internally converts to the S1(ππ*, La) state at times shorterthan their instrument response. It was further shown that the steady-state fluorescence spectra and quantum yields depend on theexcitation wavelength, while fluorescence decays were wavelength-dependent. These results were interpreted as fluorescence fromdGMP emanating from two different regions on the S1(ππ*, La)potential energy surface: one close to the Franck-Condon regionand the other along the reaction path leading toward the CI thatconnects the S1(ππ*, La) state to the S0 state, as previously pro-posed for GMP in aqueous solutions.33

Strikingly, our results show that the steady-state and time-resolved photophysics of 6tGuo are significantly different fromthat of the DNA/RNA guanine monomers in solution. Thedifferences in the steady-state absorption spectrum and verticalexcitation energies between 6tGuo and the guanine monomersprovide hints that this should be the case. In 6tGuo the red-mostabsorption band is located at ∼340 nm, while in the guaninemonomers it appears around 250 nm in aqueous solution.Comparing vertical excitation energies at the TD-PBE0 level oftheory for 9MG33 and 6tGuo shows that the S(nOπ*) is higher inenergy than the S1,2(ππ*) in 9MG, while the S(nSπ*) has thelowest energy in 6tGuo followed by the S2(ππ*, La) state(Table 1). Hence, replacement of the oxygen atom in 9MG bya sulfur atom in 6tGuo results in a remarkable change in theenergy and order of the excited states in the Franck-Condonregion of the potential energy surfaces. This is to be expectedas the CdS bond is weaker than the CdO bond, and the excitedelectronic states are typically found at lower energies in thyo-carbonyls.66

A note of caution should be given at this point as thecalculations for 9MG33 by Karunakaran et al.33 include explicitsolvent effects while those presented here for 6tGuo includeonly bulk solvation. However, we note that our previous workwith 4tThd shows that inclusion of two water molecules in thefirst solvation shell does not appreciably change the energy orstate ordering in the singlet and triplet manifolds.19 This is notnecessarily surprising as carbonyl groups are expected to formstronger hydrogen bonding interactions with water moleculesthan thiocarbonyl groups. In line with this expectation, the red-most absorption band of 6tGuo (Figure 1) and 4tThd20 it is notperturbed significantly in acetonitrile versus aqueous solutions.In addition, the use of a methyl group at the 9N position in 9MGversus a ribose sugar in 6tGuo can have a small but noticeableeffect on the energies of the electronic states in the Franck-Condon region.67

The transient absorption results presented in this work clearlyshow that the excited state dynamics of 6tGuo is dramaticallydifferent from that of GMP33 in solution. Excitation of GMP at267 nm results in ultrafast internal conversion from the S1 state tothe ground state, while excitation of 6tGuo at 340 nm resultsprimarily in ultrafast population of the triplet state in high yield.This can be understood in terms of the 2.5-fold higher spin-orbit coupling of the sulfur versus oxygen atom,66,68 which isexpected to significantly increase the intersystem crossing prob-ability to the triplet manifold in 6tGuo versus GMP. Hence, asshown previously in 4tThd,19,20,69 the sulfur atom promotes anintramolecular heavy-atom effect in 6tGuo, increasing the prob-ability of singlet to triplet population transfer from close to zeroin the guanine monomers to ∼0.8 in 6tGuo in solution.



The ultrafast nature of the population transfer from the singletto triplet manifold in 6tGuo can be due to the existence of a CIbetween these states, which is accessible in almost barrierlessfashion. Recently, Serrano-Andr�es and co-workers70 have locatedseveral CIs between the S1(ππ* La), S2(nOπ*), and S0 states andthe T1,2(ππ*) and T3(nOπ*) states in 9H-guanine. There is noreason a priori to why similar CIs should not be present betweenthe singlet and triplet manifolds in 6tGuo. In particular, the nearzero energy gap between the S2(ππ*, La) and the T3(nπ*) statespredicted by our calculations in water and in acetonitrile suggeststhat the ultrafast population transfer from the singlet to the tripletmanifold in 6tGuo can be explained by the presence of aCI betweenthese two states due to enhanced spin-orbit and vibronic coupling.A further distinction between the guanine monomers and

6tGuo is that population of the S(nOπ*) state in GMP33 is notobserved in solution while we propose that a small fraction ofthe population in the S2(ππ*, La) state bifurcates to the S1(nSπ*)state in 6tGuo on an ultrafast time scale. Recent quantumchemical calculations have located a CI between the S(ππ*, La)and the S(nOπ*) states and between the S(nOπ*) with the groundstate in the guanine monomers in the gas phase.54,56-59 Althoughthese CIs do not play a role in the photochemistry of 9HGua,56 weargue that analogous CIs could play a role in the photochemistry of6tGuo. Clearly, 6tGuo and 4tThd19,20,69 DNA/RNA analoguesshow significantly different excited-state dynamics compared tothe DNA/RNA monomers. In this respect, thiobases are excellentcandidates to further benchmark the predictions and accuracyof recently developed, high-level static and dynamics quantum-chemical calculations.

4. CONCLUSIONS

The excited-state dynamics of 6tGuo have been investigatedfrom the femtosecond to microsecond time scale in aqueousbuffer solution and in acetonitrile. The experimental results havebeen complemented with quantum-chemical calculations thatinclude solvent effects. It is shown that UVA excitation of 6tGuoresults in ultrafast population transfer to the triplet state in highyield characterized by a lifetime of ∼0.3 ps in aqueous solution.The triplet state is quenched by molecular oxygen (τ = 460 ns),presumably producing singlet oxygen in high yield. The tripletstate of 6tGuo decays back to the ground state in (720( 10) nsin N2-saturated solution. Hence, our results suggest that 6tGuocan act as an efficient photosensitizer when incorporated in DNAresulting in DNA photooxidative damage. This might explain thehigh phototoxicity of 6tGuo in DNA and the increased incidenceof skin cancer upon UVA light exposure in patients treated withpurine derivatives.

A second,minor decay channel is also populatedwithin 0.3 ps. Itis assigned to ultrafast internal conversion from S2(ππ*, La) to theS1(nSπ*) state. The S1(nSπ*) state repopulates the ground statein tens of picoseconds. Solvation dynamics in the triplet manifoldare argued to occur simultaneously with the S0 r S1(nSπ*)internal conversion process. Although the identification of mini-mum energy reaction pathways, CIs, and molecular dynamicssimulations are needed, the vertical excitation energies reportedin this work for 6tGuo in water and in acetonitrile support theproposed kinetic model, which is presented in Scheme 1.

After this work was accepted for publication, Gao and co-workers reported the singlet oxygen quantum yield of 6tGuo andother 6-thioguanine derivatives in aqueous solution.71 A 0.55 (0.08 singlet oxygen yield was measured for 6tGuo in aqueous tris

buffer solutions at pH 7.4 after excitation at 342 nm. The highsinglet oxygen yield reported by this group is in line with the hightriplet yield measured in this work. This further supports ourargument that 6tGuo can act as an efficient photosensitizer whenincorporated into DNA resulting in oxidative damage to thenucleic acid bases.

’AUTHOR INFORMATION

Corresponding Author*E-mail: [email protected].

’ACKNOWLEDGMENT

The authors thank theDepartment of Chemistry, CaseWesternReserve University, for support. C.E.C.-H. thanks the MississippiCenter for Supercomputer Research and theOhio SupercomputerCenter for generous allotment of computer time. C.R. thanks theDeutsche Forschungsgemeinschaft (DFG) for support.

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Excited-State Dynamics in 6-Thioguanosine from the Femtosecond to Microsecond Time Scale

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