Letter pubs.acs.org/JPCL
Excited-State Dynamics in O6‑Methylguanosine: Impact of O6‑Methylation on the Relaxation Mechanism of Guanine Monomers Brennan Ashwood,† Luis A. Ortiz-Rodríguez, and Carlos E. Crespo-Hernández* Department of Chemistry and Center for Chemical Dynamics, Case Western Reserve University, Cleveland, Ohio 44106, United States S Supporting Information *
ABSTRACT: Absorption of ultraviolet radiation by DNA bases results in ultrafast internal conversion to the ground state, which minimizes photodamage. However, exogenous and endogenous alkylating agents present in the cellular environment can methylate the nucleobases in DNA. In particular, methylation of guanosine at the O6 position in DNA leads to the formation of the O6-methylguanosine adduct, which may alter the photostability of DNA. This contribution demonstrates that O6-methylation of guanosine red shifts its ground-state absorption spectrum and slows down the rate of internal conversion to the ground state by ∼40-fold in aqueous solution. The 40-fold decrease in the rate of excitedstate decay increases the probability of photodamage within cellular DNA. It is proposed that the longer decay lifetime corresponds to relaxation of the excited-state population in O6-methylguanosine along a C6-puckered reaction coordinate in the 1ππ*(La) potential energy surface that runs parallel to an ultrafast internal conversion pathway along a C2puckered coordinate.
T
Our group has recently shown that chemical modifications at the C6 position of purine derivatives can have a large impact on the excited-state dynamics and photochemistry in DNA purine monomers.7 The initial effort focused on the photophysical, dynamical, and electronic structure differences between key tautomers of the purine derivatives,7 but the effect that O6methylation has on the excited-state dynamics and photochemistry of the Gua monomers has yet to be elucidated. In this Letter, we report the steady-state and time-resolved photophysical properties of 6MeGuo under physiological conditions. The experimental measurements are complemented with a detailed set of ground- and excited-state calculations for 6MeGuo in water. Furthermore, we report the steady-state spectra for guanosine 5′-monophosphate (GMP) and the vertical excitation energies for the enol/keto tautomers of Guo under equal conditions as those used for 6MeGuo. The results reported here are combined with previous experimental and computational studies for the Gua monomers to provide a mechanistic description of the electronic relaxation dynamics in 6MeGuo. As shown below, this strategy allows us to propose some generalizations about the electronic relaxation mechanism of the Gua monomers. Details about the chemicals and methodology used are provided in the Supporting Information (SI). Figure 1 compares the molecular structure, ground-state absorptivity, and emission spectra of 6MeGuo and GMP under equal experimental conditions of phosphate buffered saline
he absorption of UV light by DNA gives rise to a cascade of potentially mutagenic and carcinogenic biochemical processes.1 Therefore, the photophysical properties of DNA bases and their derivatives are of both fundamental and practical interest for understanding the early events in photochemical damage.2,3 Numerous studies have shown that the DNA monomers dissipate most of their excess electronic energy on an ultrafast time scale following UV excitation, making them exceptionally photostable in aqueous solution.2−6 However, seemingly small structural modifications of the canonical nucleobases can significantly affect their intrinsic photostability.5,7,8 Methylated DNA bases play a significant role in regulating gene expression and can also facilitate the formation of DNA lesions. CpG domains with 5-methylated cytosine (5mCyt) nucleotides have been shown to accelerate photodamage to DNA,9−11 and as a result, the photophysics of 5mCyt have been studied in detail.12−15 O6-methylguanosine (6MeGuo), produced from methylation of guanosine (Guo) at the O6 position in DNA by exogenous or endogenous alkylating agents,16,17 is generated in smaller amounts than 5mCyt but has been shown to exhibit a greater carcinogenic potency.18−20 The enhanced carcinogenicity of 6MeGuo originates from the structural alterations caused by O6-methylation. Specifically, O6-methylation changes the hydrogen bond properties of the guanine (Gua) nucleobase because of deprotonation at the N1 position. This leads to the inhibition of the Watson−Crick base-pairing configuration and subsequently to mispairing by DNA polymerase.21,22 If not removed by enzymatic repair,23−25 the buildup of 6MeGuo in DNA can lead to its misreading as an adenine base and mispairing with the thymine base,16,21,25 which sparks the generation of DNA mutations. © XXXX American Chemical Society
Received: August 9, 2017 Accepted: August 29, 2017 Published: August 29, 2017 4380
DOI: 10.1021/acs.jpclett.7b02090 J. Phys. Chem. Lett. 2017, 8, 4380−4385
Letter
The Journal of Physical Chemistry Letters
Table 1. Lowest Three Singlet and Triplet Vertical Excitation Energies in eV for the syn-Sugar Conformation of 6MeGuo, 6-Enolguanosine (6EnolGuo), and 6Ketoguanosine (Guo) in Water Calculated at the TD-PBE0/ IEFPCM/6-311++G(d,p)//PBE0/IEFPCM/6-311++G(d,p) Level of Theorya state
6MeGuo
6EnolGuo
Guo
ππ*, La ππ*, Lb 1 nπ* T1(ππ*) T2(ππ*) T3(ππ*)
4.72 (0.248) 5.35 (0.132) 5.42 (0.031) 3.45 4.28 4.80
4.71 (0.189) 5.38 (0.116) 5.43 (0.055) 3.45 4.27 4.84
4.91 (0.161) 5.20 (0.345) 5.48 (0.0005) 3.66 3.92 4.82
1 1
a
Oscillator strengths are provided in parentheses.
where Platt’s nomenclature is used.29 O6-methylation destabilizes the S2(ππ*, Lb) state, while it stabilizes the S1(ππ*, La) state. O6-methylation also increases the magnitude of the calculated oscillator strength for the S1(ππ*, La) transition, whereas it decreases the magnitude of the calculated oscillator strength for the S2(ππ*, Lb) transition. The predicted effect of O6-methylation on the order and strength of these electronic transitions is in very good agreement with the absorptivity spectra reported in Figure 1a. This suggests that the calculations are able to satisfactorily model the order of the electronic states in the FC region for both molecules. In particular, the calculations predict that excitation with 290 nm radiation directly populates the S1(ππ*, La) state of 6MeGuo and Guo (GMP); therefore, the emission spectra shown in Figure 1b for 6MeGuo and GMP should originate primarily from the S1(ππ*, La) state. This is supported by the optimization of the S1(ππ*, La) state of 6MeGuo in water at the TD-PBE0/IEFPCM/6-31G(d,p)//PBE0/IEFPCM/6-311+ +G(d,p) level of theory (Figure S2), which predicts an adiabatic energy of 3.73 eV (332 nm) that is in very good agreement with the emission band shown in Figure 1. Table 1 also shows that the character and order of electronic states is essentially identical for both 6MeGuo and 6EnolGuo in water, while Figure S2 shows that both molecules have very similar S1(ππ*, La) minima. This suggests that 6MeGuo has a similar electronic structure to 6EnolGuo and its corresponding enol nucleobase (6EnolGua). Hence, the results presented in this study for 6MeGuo could also serve as a suitable model compound to investigate how the solvent affects the electronic relaxation mechanism reported for 6EnolGua in the gas phase.30−33 While the keto form is the most stable tautomer of Guo or GMP in solution,27,34 the 6-enol tautomers persist in the gas phase and contribute to the overall photophysics of the Gua chromophore.30−32,34−37 Pump−probe experiments have been performed on Gua, Guo, and GMP in the gas phase,38−41 which demonstrate that the Gua chromophore decays to the ground state with biexponential kinetics in hundreds of femtoseconds, similar to what is observed in solution.26−28 However, it remains to be determined whether the enol tautomers contributed to the observed electronic relaxation dynamics in those gas-phase studies. Tautomer-selective molecular beam fluorescence experiments30 and pump−probe experiments33 have been performed for 6EnolGua in the gas phase. The authors observed a fluorescence decay process in 12−13 ns30,33 and a 40 ns decay path33 that populates a longlived dark state tentatively assigned to a triplet state. These excited-state dynamics are remarkably different than those
Figure 1. Top: Structure and standard numbering of 6MeGuo (left; R1 = methyl; R2 = ribose), 6EnolGuo (left; R1 = hydrogen; R2 = ribose), and GMP (right; R1 = hydrogen; R2 = ribose 5′-monophosphate). (a) Ground-state absorptivity and (b) emission spectra of 6MeGuo (black) and GMP (blue) in PBS at pH 7.4.
(PBS) solution at pH 7.4. 6MeGuo exhibits two low-energy absorption bands of similar strength centered at 248 (6800 M−1 cm−1) and 280 nm (6490 M−1 cm−1), while GMP exhibits overlapping absorption bands centered at 253 (9740 M−1 cm−1) and 275 nm (6392 M−1 cm−1). Another absorption band is observed at 210 nm for 6MeGuo that is shifted to higher energies in GMP. Upon excitation with 290 nm radiation, 6MeGuo exhibits a broad emission spectrum centered at 380 nm (Figure 1b), while that of GMP is centered at 360 nm and shows a tail that extends beyond 550 nm.26−28 A fluorescence quantum yield of (4.0 ± 0.5) × 10−3 was determined for 6MeGuo (see also Figure S1), which is 31-fold larger than that measured for GMP in back-to-back under the same experimental conditions (1.3 ± 0.5) × 10−4. The steadystate results demonstrate that O6-methylation modifies the photophysics of the Guo chromophore. Vertical excitation energies for the three lowest-energy singlet and triplet states of 6MeGuo, 6-enolguanosine (6EnolGuo), and Guo were calculated in water to elucidate the order of the electronic states in the Frack−Condon (FC) region (Table 1). Guo was used as a model compound of GMP in these calculations because the phosphate group is not expected to drastically affect the vertical excitation energies. This is supported by transient absorption studies in which Guo and GMP undergo identical excited-state decay kinetics in aqueous solution.28 Table 1 presents the results for the synsugar conformation of all three Gua derivatives, in which the O6-methyl/hydrogen is pointing toward N1 in 6MeGuo and 6EnolGuo. Calculations presented in the SI predict that these are the lowest-energy ground-state structures in water (Tables S1−S3). Results for the anti-sugar conformations, and for the other rotamers of 6MeGuo and 6EnolGuo, are presented in Tables S4−S13. According to these calculations, the lowestenergy absorption bands for both 6MeGuo and GMP correspond to the S2(ππ*, Lb) and S1(ππ*, La) transitions, 4381
DOI: 10.1021/acs.jpclett.7b02090 J. Phys. Chem. Lett. 2017, 8, 4380−4385
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Figure 3. (a) Representative kinetic decay traces of 6MeGuo at select probe wavelengths. Traces from the UV (330−390 nm) and visible (400−700 nm) bands were globally fit separately to a two-component sequential model. (b) DAS extracted from the global analysis.
Figure 2. Transient absorption spectra of 6MeGuo in PBS at pH 7.4 following excitation at 290 nm. (a) Dynamics during the crosscorrelation of the pump and probe beams; (b) dynamics during the initial ∼3 ps time delay; and (c) dynamics during the next tens of picoseconds time window. The region from 575 to 585 nm is omitted due to Rayleigh scattering from the overtone of the pump beam.
varies with probe wavelength (Figure S5a). An average value of 2.4 ± 1.6 ps was obtained for this lifetime from global analysis of the UV-only probe region. The first lifetime associated with the visible band decays faster, in 0.95 ± 0.07 ps, and only the blue side of this band (410−510 nm) exhibits a moderate dependence on the probe wavelength (Figure S5b). Conversely, the second lifetime extracted from the individual global analyses of the UV and visible regions is identical within experimental uncertainties, 41 ± 2 and 37 ± 2 ps, respectively. Thus, this latter lifetime is likely associated with a single relaxation process. Best-fit curves of representative decay traces and DAS obtained from this analysis are shown in Figure 3. According to the vertical energies, excitation of 6MeGuo at 290 nm directly populates the S1(ππ*, La) state in aqueous solution. It is proposed that excitation of 6MeGuo at 290 nm results in ultrafast branching of the population that follows two parallel reaction coordinates, as depicted in Scheme 1. The first
observed for the keto tautomers of the Gua derivatives, and an investigation of the excited-state dynamics of 6MeGuo in solution can also provide important insights into the mechanistic differences between enol and keto-Gua monomers. Femtosecond transient absorption spectroscopy was used to investigate the excited-state relaxation mechanism of 6MeGuo upon excitation at 290 nm. Excitation of 6MeGuo results in the formation of two broad transient absorption bands within the cross-correlation of the pump and probe beams (Figure 2a), which are centered at 610 and below 350 nm at a delay of ∼0.38 ps. Their intensity continues to rise during the next ∼2 ps, while the visible band significantly narrows and its maximum blue shifts to 595 nm (Figure 2b). A uniform decay across the probe window in tens of picoseconds follows this process until no absorption signal is detected at delays longer than ∼130 ps (Figure 2c). Initially, we globally fit the time domain data across the entire probe window with a two-component sequential kinetic model, yielding lifetimes of 1.8 ± 0.2 and 41.7 ± 0.5 ps. Best-fit curves to representative decay traces and corresponding decayassociated spectra (DAS), obtained using this global analysis, are shown in Figure S3. A closer inspection of the transient absorption data reveals, however, that the dynamics of the UV (330−390 nm) and visible (>410 nm) absorption bands are not identical during the initial ∼10 ps delay (Figure S4). This suggests that an additional pathway is available. In order to extract this pathway, we divided the spectral probe windows in two regions and globally fit each one separately also using a two-component kinetic model. Clearly, this latter analysis leads to two decay lifetimes for each probe region. The first of the two lifetimes associated with the dynamics of the UV band
Scheme 1. Proposed Relaxation Mechanism of 6MeGuo in Aqueous Solution
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DOI: 10.1021/acs.jpclett.7b02090 J. Phys. Chem. Lett. 2017, 8, 4380−4385
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emission spectra shown in Figure 1b for 6MeGuo (380 nm) and GMP (360 nm) demonstrate that the emissive S1(ππ*, La) minimum is lower in energy in 6MeGuo than that in GMP. O6-methylation slows down IC to the S0 state for a large fraction of the S1(ππ*, La) population as 6MeGuo undergoes decay to the ground state in ∼40 ps, in addition to the ∼1 ps IC, which is also observed in the Gua monomers in aqueous solution.26−28,43 Similar to 6EnolGua,31,32 we propose that 6MeGuo primarily undergoes relaxation through the C6 reaction coordinate. Hence, it is shown that replacing the H atom at the C6 position of Gua with a O, OH, or OMe can significantly affect the topology of the electronic surfaces, regulating the access to key CIs that control the relaxation of electronic energy to the S0 state, as proposed for other purine derivatives. 7 C6-functionalization in turn enhances the relaxation of electronic energy through the C6 reaction coordinate in solution, while the C2 reaction coordinate plays a secondary but important role, depending on the nature of the substitution.7 6MeGuo is a naturally occurring DNA base derivative with both fundamental and biological interest. It remains to be determined whether O6-methylation of Guo increases the photoreactivity of the Gua nucleobase in DNA. We have shown that 6MeGuo absorbs radiation at lower energies than GMP with larger absorptivity coefficients in the UVB spectral region, which increases the probability to absorb sunlight. Furthermore, UVB excitation of 6MeGuo traps a significant fraction of the excited-state population in the S1(ππ*, La) potential energy surface for ∼40 ps before repopulation of the ground state can occur. The 40-fold increase in the S1(ππ*, La) lifetime compared to Guo or GMP increases the probability that 6MeGuo can react with adjacent base-paired or base-stacked nucleobases in cellular DNA. As such, this contribution provides new mechanistic insights of direct relevance to the excited-state dynamics and photochemistry of O6-methylated Guo in DNA.
pathway is associated with C2 puckering and pyramidalization of the amino group (C2 reaction coordinate, hereafter), while the second pathway is associated with bending of the O6methyl group and pyramidalization of the C6 atom (C6 reaction coordinate), in analogy with calculations for 6EnolGua (see also the optimized S1(ππ*, La) minimum in Figure S2).31,32 Recent experimental and computational results for purine derivatives7 and Gua42 in aqueous solutions support the proposal of an initial parallel relaxation mechanism involving both the C2 and C6 reaction coordinates, where the latter coordinate plays a larger role in solution. The population following the C2 reaction coordinate is proposed to reach a conical intersection (CI) with the ground state, (S1/S0)CI1, which repopulates the ground state with excess vibrational energy. Access to (S1/S0)CI1 is associated with the τ1 extracted from analysis of the visible transient absorption band (vide supra). Vibrational cooling (VC) then ensues in the ground state, which is associated with the first lifetime (τ2 in Scheme 1) extracted from analysis of the UV transient absorption band. We envision this relaxation pathway to be analogous to that originally proposed by Karunakaran et al.27 and more recently supported by Lee et al.43 when investigating the dynamics of GMP in aqueous solution. Although the results presented in this Letter do not show any clear evidence of the population of a S 1 (ππ*, L a ) minimum across this shallow reaction coordinate,27,43 the ∼1 ps lifetime associated with pathways lends some support to such a prospect. The population that follows the C6 reaction coordinate is proposed to reach a minimum in the S1(ππ*, La) potential energy surface before surmounting an energy barrier to access a second CI with the ground state, (S1/S0)CI2, as depicted in Scheme 1. Given the single bond type between carbon and the OMe or OH functional group at the C6 position in 6MeGuo or 6EnolGuo, respectively, out-of-plane motion along the C6 reaction coordinate should be more favorable in these purine derivatives than that in Guo. The population of a S1(ππ*, La) minimum along the C6 reaction coordinate is further supported by the S1-optimized minimum presented in Figure S2. The tens of picoseconds lifetime (τ3) extracted from both the UV and visible probe regions is associated with this relaxation pathway. We envision the S1(ππ*, La) minimum and the CI associated with this decay to be analogous to those calculated by Chen31 and Marian32 for 6EnolGua. The population following the C6 reaction coordinate can either fluoresce from the S1(ππ*, La) minimum or surmount an energy barrier to internally convert to the S0 state through the (S1/S0)CI2, as depicted in Scheme 1. The fluorescence quantum yield of 0.4% reported above indicates that the majority of ground-state repopulation occurs nonradiatively through internal conversion (IC). The presence of a methyl group in 6MeGuo instead of a hydrogen atom in 6EnolGua,31,32 or an oxygen atom in Gua,42 can explain the 30fold higher fluorescence yield measured for 6MeGuo compared to that for GMP under equal experimental conditions. It is suggested that O6-methylation raises the energy barrier to (S1/ S0)CI2, which increases the time required for IC following vibrational relaxation along the C6 reaction coordinate. This is supported by previous quantum calculations, which indicate that the (S1/S0)CI near the S1 minimum of H9-EnolGua is ∼0.35 eV higher in energy than for the H7- or H9-keto tautomer of Gua.32 Additionally, calculations at the CASPT2 level of theory predict that the energy gap between the S1 minimum and the S0 state is ∼2 eV larger for H9-6EnolGua than that for the H7- and H9-keto tautomers.31 The maxima of
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b02090. Experimental and computational methods; steady-state excitation spectra; representative decay traces with single global analysis across the probe region and normalized decay traces; DFT-optimized geometries and TD-DFT vertical excitation energies and Kohn−Sham orbitals for each conformation of 6MeGuo, 6EnolGuo, and Guo; and XYZ coordinates for the optimized S0 and S1 state geometries (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Brennan Ashwood: 0000-0001-7614-6071 Carlos E. Crespo-Hernández: 0000-0002-3594-0890 Present Address †
B.A.: Department of Chemistry, Institute for Biophysical Dynamics, and James Franck Institute, The University of Chicago, Chicago, IL 60637 U.S.A. 4383
DOI: 10.1021/acs.jpclett.7b02090 J. Phys. Chem. Lett. 2017, 8, 4380−4385
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge funding from the National Science Foundation (Grant # CHE-1255084 and CHE-1539808). This work made use of the High Performance Computing Resource in the Core Facility for Advanced Research Computing at CWRU.
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DOI: 10.1021/acs.jpclett.7b02090 J. Phys. Chem. Lett. 2017, 8, 4380−4385
Letter
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DOI: 10.1021/acs.jpclett.7b02090 J. Phys. Chem. Lett. 2017, 8, 4380−4385
